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. 2024 Feb 7;7(3):667–679. doi: 10.1021/acsptsci.3c00272

Investigating LIM (Lin-11, Isl-1, and Mec-3) Kinases and Their Correlation with Pathological Events and Microtubule Dynamics in an Experimental Model of Spinal Cord Injury in Rats

Abhishek Roy 1, Zarna Pathak 1, Hemant Kumar 1,*
PMCID: PMC10928889  PMID: 38481685

Abstract

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The spinal cord injury (SCI) and the neurodegenerative processes accompanying it follow an intricate pathway with very limited options for treatment strategies until now. Microtubules, essential for the growth and maintenance of neurons, are mostly disorganized and destabilized due to neurodegeneration. Regeneration or plasticity is restricted to the adult central nervous system (CNS) due to several intrinsic and extrinsic mechanisms. Some fundamental or injury-induced expressions of specific molecules can be inhibited or antagonized pharmacologically to protect neurons to a certain extent after neurodegeneration. Accordingly, these molecules offer an excellent target as a therapeutic approach to promote neuroprotection. LIM kinases (LIMKs) are one of these molecules that phosphorylates members of the actin-depolymerizing factor (ADF)/cofilin family of actin-binding and filament-severing proteins. The individual role of LIMKs has not yet been studied in the pathology of SCI. In this study, we targeted LIMK and checked its role in microtubule destabilization in vitro. LIMK1 was found to be upregulated after microtubule depolymerization and inhibition of LIMK with specific inhibitor-protected neurons. Then, we checked the expressions of individual LIMKs throughout different time points across SCI in a rat contusion model, correlating with established pathophysiological markers. The phosphorylated form of LIMK1 was found to be elevated at chronic time points after injury, where scar formation and diminution of neurons prevail. Finally, we targeted the LIMK pathway with its specific inhibitor BMS-5, which showed neuroprotection after SCI. Overall, our results provided a concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat SCI-associated neurodegeneration.

Keywords: spinal cord injury, microtubules, LIM kinase, regeneration, cofilin, BMS-5

A complex and finely organized neural network of billions of neurons is required for the nervous system to operate appropriately.1 The cytoskeleton sustains the characteristic highly polarized shape of neurons and helps govern neuronal mobility. As a result, coordinating cytoskeleton remodeling is critical for permitting substantial changes in neuronal morphology.2 The ability of neurons to direct their axons along suitable paths is necessary to form an adequately linked and subsequently functioning nervous system. At the tip of the developing axon, there is a dynamic, sensory-motile structure called the growth cone, which directs the axon and, hence, neuronal development. Growth cones contain two major cytoskeletal filaments, actin filaments, and microtubules, ensuring neuronal process growth.3 Traumatic SCI causes the persistent loss of motor and sensory functions due to alterations in the extrinsic microenvironment and intrinsic neuronal properties, which limit plasticity and neuronal regeneration.4 After the primary injury, a long-term secondary injury is perceived as a complex and multifactorial stage that may result in many detrimental consequences, including oxidative stress, inflammation, and mitochondrial dysfunction, which all contribute to neuronal death and restrict recovery.5,6 Reactive astrocytes, neural/glial antigen 2 (NG2) glia, and microglia proliferate after SCI, forming a barrier surrounding the epicenter. The core of this barrier is made up of non-neural stromal cells and components of the extracellular matrix (ECM), such as fibronectins, collagens, proteoglycans, and laminins.7

The LIMK family of serine protein kinases, which includes LIMK1 and 2, are the central regulators of cytoskeletal dynamics.8 LIMK proteins are differentially expressed in various tissues; their expression is controlled differently, distinct signaling pathways trigger them, and their activity is regulated accordingly.9 While LIMK1 is activated by the Rho/Rho-associated coiled-coil containing protein kinase (ROCK1 and 2) and Rac/p-21-activated kinase (PAK1, 2, and 4) pathway,10,11 LIMK2 is preferentially activated by the ROCK pathway.12,13 LIMK1 is majorly expressed in the nervous system; it is concentrated in presynaptic terminals during synapse maturation, suggesting that LIMK1 regulates activities of the nerve terminal that change during the maturational process.14 Knockout study revealed that the LIMK1 knockout mice developed normally and were fertile, but they had abnormalities in dendritic spine morphology and synaptic function.15 On the other hand, the only phenotypic abnormality seen in LIMK2-deficient mice occurred during spermatogenesis in the testes. The testes of those mice were smaller, and some of the spermatogenic cells in the seminiferous tubules were partially degenerated.16 LIMK1 is activated upon phosphorylation of threonine (Thr-) 508 by ROCK, the protein downstream of a pathway that inhibits axon growth and sprouting.17 Mitotic phosphorylation of LIMK1 at Thr-508 in its kinase domain causes it to separate from microtubules and migrate from the prometaphase to the metaphase centrosome, where it phosphorylates and inactivates the actin-depolymerizing protein cofilin.18 Cofilin controls actin dynamics by splitting actin filaments and removing the actin monomer from the pointed end of actin filaments.19 ADF/cofilin knockdown significantly inhibited NGF-induced neurite extension in PC12 cells.20 The significance of actin dynamics in the regeneration of adult mouse dorsal root ganglia (DRG) neurons was investigated in a study by Tedeschi et al. In a process known as “conditioning”, these neurons rebuilt their central axon when their peripheral axon was damaged prior to CNS injury. Their study revealed that activation of ADF/cofilin underlying improved growth cone dynamics is crucial for regenerative conditioned growth.21 Cofilin can no longer bind to actin after being phosphorylated on serine 3 by LIMK1, prompting the aggregation of actin polymers. As a result, actin cannot interact with microtubules, and microtubules become depolymerized to a large extent. In contrast to a growth cone-like structure, injured mature CNS neurons form a dystrophic bulb called a retraction bulb, which lacks regenerative capacity.22 LIMK1 expression was found to enhance cofilin phosphorylation while suppressing growth cone motility and extension in a kinase-dependent manner in chick DRG neurons. In the same study, the phosphorylation site of cofilin Ser-3 was mutated to the nonphosphorylatable alanine and this cofilin (S3A) was employed as a constitutively active form. Coexpression of S3A significantly restored the suppressive impact of LIMK1, demonstrating that ectopically expressed LIMK1 limits growth cone motility and extension principally via the phosphorylation and inactivation of cellular cofilin.23 Moreover, the cofilin/LIMK1 pathway was found to be involved in the formation of spinal motor circuitry and regeneration of the sciatic nerve. The adult mouse spinal cord had a significantly lower amount of phosphorylated (p) cofilin in comparison to the postnatal stages. Nevertheless, following a peripheral nerve injury, p-cofilin levels at the lesion site increased rapidly in a LIMK1-dependent way.24

Herein, we checked the differential expression of phosphorylated LIMK in SCI-associated neurodegeneration and its correlation with major glial and neuronal markers. For this purpose, we investigated the expression of LIMK and the effect of its inhibition on microtubule dynamics and neurite outgrowth in Neuro2A cells. Then, we checked the expressions of LIMKs in a rat contusion model of SCI. After establishing the expression pattern of phosphorylated LIMKs, again we checked the effects of LIMK inhibition using a commercially available inhibitor, BMS-5/LIMKi-3, in axonal regeneration or sprouting.

Results

Altered Expression of Phosphorylated LIMK through Microtubule Depolymerization in Neuro2A Cells and Effect of LIMK Inhibition in Regulating Microtubule Dynamics

We checked the expression of phosphorylated LIMK1 through microtubule depolymerization in vitro and its modulatory effect on neuroprotection in Neuro2A cells. Nocodazole becomes attached to free tubulin and inhibits it from being incorporated into microtubules, causing microtubule depolymerization.25 As obtained from the results of a previous study conducted by Naomi S. Hachiya et al.,26 nocodazole at a concentration of 30 μM for 30 min was found to depolymerize microtubules in neuro2A cells. We chose nocodazole, as it simulated neurodegeneration through its microtubule depolymerizing activity. Nocodazole was used at a concentration of 30 μM and was found to be responsible for microtubule depolymerization through a significant upregulation in the expression of phosphorylated LIMK1. We checked the effect of LIMK inhibition with an inhibitor BMS-5 in both the presence and absence of nocodazole. The cell viability of BMS-5 was determined by using different concentrations. Cells were viable at 1, 2, 5, and 10 μM concentrations and further analyzed for the inhibitory potential of phosphorylated LIMK and its downstream protein, phosphorylated cofilin, by Western blot analysis. We found that at 5 μM concentration of BMS-5, there was a significant downregulation of phosphorylated LIMK1 and cofilin compared to the control [Figure 1A–C]. Hence, a 5 μM concentration of BMS-5 was used further to check its effect on neurite growth. BMS-5 alone was found to cause a significant downregulation of phosphorylated LIMK1 as compared to the nocodazole group, while BMS-5 combined with nocodazole was also responsible for a reduction in phosphorylated LIMK1 when compared to nocodazole [Figure 1D,E]. We also checked the expressions of β-tubulin III and KIF5B for the same groups. Here, the nocodazole-treated group inhibited neurite outgrowth; hence, a downregulation in the expressions of both these proteins was observed. Concurrently, the BMS-5 alone group was found to cause a significant increase in both these proteins as compared to the nocodazole group. However, in the case of the combined group, though an upregulation was observed, it was not statistically significant [Figure 1D,F,G].

Figure 1.

Figure 1

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Nocodazole-induced phosphorylation of LIMK through microtubule depolymerization in Neuro2A cells and inhibition of LIMK potentiated neurite growth. (A) Representative Western blot images of phosphorylated LIMK1 and cofilin for control and different concentrations of BMS-5. (B, C) Quantification of phosphorylated LIMK1 and phosphorylated cofilin, respectively. (D) Representative Western blot images of phosphorylated LIMK1, β tubulin III, and KIF5B for control, nocodazole, BMS-5, and BMS-5 combined with nocodazole. (E–G) Quantification of phosphorylated LIMK1, β tubulin III, and KIF5B, respectively. ***p < 0.001 vs control, **p < 0.01 vs control, *p < 0.05 vs control, ###p < 0.001 vs nocodazole, ##p < 0.01 vs nocodazole, and #p < 0.05 vs nocodazole. Data is represented as mean ± SEM (n = 3).

LIMK Inhibition Promotes Neurite Outgrowth through the Stabilization of Tyrosinated Tubulin

We designed our experiment into six different groups, namely, control, nocodazole, BMS-5, epothilone A, epothilone A combined with nocodazole, and BMS-5 combined with nocodazole [Figure 2A]. Here, epothilone A at a concentration of 10 nM was used as a positive control for microtubule stabilization. Nocodazole and BMS-5 were used at a concentration of 30 and 5 μM, respectively. The destabilization or stabilization of the microtubule network can be differentiated using particular tyrosinated and detyrosinated tubulin antibodies.27 Microtubules enriched in detyrosinated tubulin are found to be localized mainly in the axon and dendritic processes of neurons and stable to nocodazole treatment, whereas microtubules containing tyrosinated tubulin have a widespread intracellular distribution and become depolymerized completely with the treatment of nocodazole.28 We found that the nocodazole-treated group showed a significant reduction in neurite growth compared to the control group as evidenced by less tyrosinated tubulin [Figure 2B]. Both BMS-5 and BMS-5 combined with nocodazole showed a significant enhancement in neurite outgrowth compared to the nocodazole group alone [Figure 2C]. Surprisingly, we found that both BMS-5 and epothilone A-treated groups showed almost the same elevation in neurite outgrowth as compared to the nocodazole. It has also been found that BMS-5 combined with nocodazole showed more neurite outgrowth as compared to the group where epothilone A was combined with nocodazole [Figure 2C].

Figure 2.

Figure 2

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LIMK inhibition promoted neurite outgrowth through the stabilization of microtubules. (A) Representative images of immunocytochemistry showing detyrosinated tubulin (green), tyrosinated tubulin (pink), and phalloidin (yellow) along with DAPI (blue) for different groups, namely, control, nocodazole, BMS-5, epothilone A, epothilone A combined with nocodazole, and BMS-5 combined with nocodazole in Neuro2A cells (2–3 fields/slide, n = 3/group) (scale bar, 200 μm). (B) The corrected total cell fluorescence (CTCF) for tyrosinated tubulin in different groups. (C) Graph showing neurite growth (μm) in the aforementioned groups. *p < 0.05 vs control, ****p < 0.0001 vs control, ###p < 0.001 vs nocodazole, ##p < 0.01 vs nocodazole. Data is represented as mean ± SEM (n = 3).

SCI Induces an Upregulation in the Expression of Phosphorylated LIMK1 at Chronic Time Points of Injury and Its Correlation with Major Pathophysiological Markers

We checked the expression of phosphorylated LIMK1 and its major substrate cofilin at different time points starting from day post injury (DPI) 1, 7, 14, 21, and 28 [Figure 3A]. We found that there was a significant increase in phosphorylated LIMK1 and cofilin at DPI-14 and DPI-28 as compared to sham [Figure 3D,E]. Furthermore, we checked the expression of phosphorylated LIMK2, but here, we did not find any significant changes at DPI-14, 21, and 28, the later time points of injury [Figure 3F]. To confirm the findings from Western blotting, we checked the expression of phosphorylated LIMK1 by immunohistochemistry. Here, we also found a significant upregulation as compared to sham [Figure 3C,G]. Further, we checked the expressions of major glial, neuronal, tight junctions, and microtubule-specific markers. A time response curve was obtained along with different time points starting from DPI-1 to 28 [Figure 4A]. The major intermediate filament protein glial fibrillary acidic protein (GFAP) was found to be significantly upregulated at later time points, suggesting the formation of glial scar consisting of reactive astrocytes at the borders of the injury epicenter.29 Phosphorylation of myosin light-chain phosphatase (MLCP), another substrate of ROCK, is increased by the growth inhibitory substances present in a glial scar, ultimately leading to growth cone collapse.30 This finding correlates to the observation of our study where an increased expression in major glial scar forming protein GFAP corresponds to an elevation in the phosphorylation of another ROCK substrate, LIMK31 [Figure 4B]. Next, we checked the expression pattern of class III β tubulin, which is recognized as one of the first proteins in mammalian and avian development that is associated with the cytoskeleton of neurons and it is noteworthy to say that the human class III β tubulin protein is 99.11% similar to the rat β III.32 In a previous study, the overexpression of LIMK1 in chick DRG neurons led to a reduction in the growth of neurites and alterations in the morphology of axonal growth cones.23 Hence, an increased expression of LIMK1 with the decline of β tubulin III confirms the impediment of neurite growth as time progresses [Figure 4C]. It has been reported that the phosphorylation and inactivation of cofilin are caused by the polarity protein Par-3 deficiency in MDCK cells, and this reduced cofilin activity contributed to defects in tight junction assembly.33 In support of this view, we found significant downregulation in the expression of occludin at DPI-28 [Figure 4D]. To investigate the destabilization of microtubules, we checked the expression of KIF5B, an ATP-driven motor protein, involved in anterograde transport along microtubules.34 KIF5B was found to be downregulated in a time-dependent manner, and significant downregulation occurred at DPI-28 [Figure 4E], suggesting that an upregulation of phosphorylated LIMK1 is associated with an impairment in the transport mechanism. Hence, the activation of LIMK1 was found to aggravate the formation of scar and the associated diminution of neurons.

Figure 3.

Figure 3

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SCI caused phosphorylation of LIMK1 and its immediate downstream protein cofilin at later time points of injury. (A) Representative Western blot images of phosphorylated LIMK1, LIMK1, phosphorylated cofilin, cofilin, and phosphorylated LIMK2 for sham, DPI-1, 7, 14, 21, and 28 in spinal cord tissue (n = 3/time point). (B) The reference axis showing rostral (R), caudal (C), epicenter (E), medial (M), and lateral (L) regions in the longitudinal image of the injured spinal cord. The epicenter region is further analyzed in panel C by immunohistochemistry. (C) Representative immunohistochemistry images of phosphorylated LIMK1 for sham, DPI-14, and DPI-28 in spinal cord tissue (2–3 fields/slide, n = 3/group) (scale bar, 50 μm). (D–F) Quantification of phosphorylated LIMK1, phosphorylated cofilin, and phosphorylated LIMK2, respectively. (G) Fluorescence intensity quantification of phosphorylated LIMK1. **p < 0.01 vs sham, *p < 0.05 vs sham. Data is represented as mean ± SEM.

Figure 4.

Figure 4

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SCI-induced changes in the expressions of major pathophysiological markers GFAP, β tubulin III, occludin, and KIF5B. (A) Representative Western blot images of GFAP, β tubulin III, occludin, and KIF5B for sham, DPI-1, 7, 14, 21, and 28 in spinal cord tissues. (B–E) Quantification of GFAP, β tubulin III, occludin, and KIF5B, respectively. **p < 0.01 vs sham, *p < 0.05 vs sham. Data is represented as mean ± SEM (n = 3).

Activation of LIMK in SCI Induces ECM Remodeling, Neuronal Loss, and Glial Activation at Chronic Phases of Injury

We checked the expressions of the major ECM protein laminin at DPI-14 and DPI-28 through immunohistochemistry [Figure 5B]. Significant accumulation of laminin at DPI-14 and DPI-28 suggests the disorganization of ECM at the epicenter of injury. The stimulation of small GTPases and their downstream proteins enhances cellular adhesion to collagen, fibronectin, and laminin, and this was evident from our finding with an increased expression of laminin.35 We also checked the expressions of GFAP and β tubulin III at these particular time points [Figure 5C]. Here, a major upregulation in GFAP and downregulation in β tubulin III justify the findings obtained from Western blot.

Figure 5.

Figure 5

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Activation of LIMK leads to the accumulation of laminin, downregulation of β tubulin III, and upregulation of GFAP at chronic phases of injury. (A) The reference axis showing rostral (R), caudal (C), epicenter (E), medial (M), and lateral (L) regions in the longitudinal image of the injured spinal cord. The epicenter region is further analyzed in panels (B) and (C) by immunohistochemistry. (B) Representative immunohistochemistry images of laminin for sham, DPI-14, and 28 in spinal cord tissue (2–3 fields/slide, n = 3/group). (C) Representative immunohistochemistry images of GFAP and β tubulin III for sham, DPI-14, and 28 in spinal cord tissue (2–3 fields/slide, n = 3/group) (scale bar −50 μm). (D, E) Fluorescence intensity quantification of laminin, GFAP, and β tubulin III, respectively. *** p < 0.001 vs sham, **p < 0.01 vs sham, and *p < 0.05 vs sham. Data is represented as mean ± SEM.

LIMK Inhibition Corresponds to Significant Changes in the Expressions of Glial, Neuronal, and Microtubule Markers

As we found the most significant upregulation of phosphorylated LIMK1 in DPI-28, we chose this particular time point for the inhibition and to investigate the changes in the expressions of glial, neuronal, and microtubule markers along with the inhibition [Figure 6A]. As the inhibitor, BMS-5 is specific to LIMK, first, we checked the expression of phosphorylated LIMK1 with two different single doses of BMS-5, 1 h post injury. The higher dose (1 mg/kg) was found to be more effective in downregulating the expression of both phosphorylated LIMK1 and cofilin significantly than the lower dose (0.3 mg/kg) as compared to the vehicle group [Figure 6B,C]. While checking the effect in glial, neuronal, and microtubule markers, we found a significant decrease in the expression of GFAP and simultaneously a significant upregulation in both β tubulin III and KIF5B as compared to vehicle with a dose of 1 mg/kg BMS-5 [Figure 6D–F].

Figure 6.

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LIMK inhibition corresponds to significant changes in the expressions of GFAP, β tubulin III, and KIF5B. (A) Representative Western blot images of phosphorylated LIMK1, phosphorylated cofilin, GFAP, β tubulin III, and KIF5B for sham, vehicle, BMS-5 (0.3 mg/kg), and BMS-5 (1 mg/kg) in spinal cord tissue. (B–F) Quantification of phosphorylated LIMK1, phosphorylated cofilin, GFAP, β tubulin III, and KIF5B, respectively. ***p < 0.001 vs sham, **p < 0.01 vs sham, *p < 0.05 vs sham. ##p < 0.01 vs vehicle, and #p < 0.05 vs vehicle. Data is represented as mean ± SEM (n = 3).

Inhibition of LIMK Modulates Fibrotic Scar, Attenuates Glial Scar, and Promotes Neuroprotection through Functional Recovery

We colocalized GFAP and collagen IV for vehicle and treatment groups and measured the lesion area [Figure 7A,E]. Inhibition of BMS-5 was found to reduce the lesion area compared to the injury group. Then, we checked the effect of BMS-5 for different markers. Both 0.3 and 1 mg/kg of BMS-5 were found to attenuate fibrotic scar formation as shown by the immunohistochemistry of laminin [Figure 7B,F]. As a consequence of SCI, activated microglia shift their orientation to the lesion, engulfing and phagocytosing cellular debris. We checked the microglial condition in the injury and treated groups through the expression of ionized calcium binding adaptor molecule 1 (IBA1). An immunohistochemistry of the injured spinal cord showed elevated levels of IBA1 [Figure 7C,G]. The small aggregates of IBA1 displayed a globoid cytoplasmic appearance rather than the normal ramified morphology. This finding was consistent with the study by Miller et al.36 Inhibition of LIMK was found to reduce IBA1 expression, suggesting the effect of BMS-5 in preventing chronic microglia-mediated inflammation37 [Figure 7C,G]. Along with this, the dose of 1 mg/kg BMS-5 was found to be more effective in fostering neuroprotection and alleviating glial scar formation [Figure 7D,H]. We also confirmed this finding from qRT-PCR. 1 mg/kg dose of BMS-5 was found to promote neuroprotection as it showed upregulated expressions of neuronal marker growth-associated protein 43 (GAP-43) and β tubulin III [Figure 7I,J]. In addition, BMS-5 was found to downregulate the major microglial marker integrin subunit α M (Itgam) as compared to the vehicle group [Figure 7K]. Further, we studied the effect of BMS-5 using BBB score in sham, vehicle, and BMS-5-treated groups at DPI-1, 7, 14, 21, and 28. SCI animals exhibited paraplegia, which correlates to a BBB score of 0 on DPI-1 in both the vehicle and BMS-5-treated groups. The BBB score then improved dramatically after DPI-14 and continued to improve until DPI-28 in the BMS-5-treated groups. The sham group had the highest BBB score of 21 [Figure 7L].

Figure 7.

Figure 7

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Inhibition of LIMK reduces lesion area, modulates the expressions of laminin, GFAP, and β tubulin III, and promotes neuroprotection through functional recovery. (A) Representative immunohistochemistry images of GFAP and collagen IV for vehicle, BMS-5 (0.3 mg/kg), and BMS-5 (1 mg/kg) in full longitudinal sections of spinal cord tissue (scale bar, 200 μm) (n = 3/group). (B) Representative immunohistochemistry images of laminin for sham, vehicle, BMS-5 (0.3 mg/kg), and BMS-5 (1 mg/kg) in spinal cord tissue (2–3 fields/slide, n = 3/group) (scale bar, 50 μm). (C) Representative immunohistochemistry images of IBA1 for sham, vehicle, BMS-5 (0.3 mg/kg), and BMS-5 (1 mg/kg) in spinal cord tissue (n = 3/group) (scale bar, 50 μm). (D) Representative immunohistochemistry images of GFAP and β tubulin III for sham, vehicle, BMS-5 (0.3 mg/kg), and BMS-5 (1 mg/kg) in spinal cord tissue (2–3 fields/slide, n = 3/group) (scale bar, 50 μm). (E) Graph showing lesion area measurement (mm2) for vehicle and both treatment groups. (F–H) Fluorescence intensity quantification of laminin, IBA1, GFAP, and β tubulin III, respectively. (I–K) mRNA expressions of GAP-43, β tubulin III, and Itgam, respectively, in sham, vehicle, and BMS-5 (0.3 and 1 mg/kg) treatment groups (n = 3). (L) Functional recovery was assessed in open-field testing by using the 21-point Basso, Beattie, and Bresnahan (BBB) locomotor test at 1, 7, 14, 21, and 28 days after SCI [n = 6/group]. ****p < 0.0001 vs sham, *** p < 0.001 vs sham, **p < 0.01 vs sham, ####p < 0.0001 vs vehicle, ###p < 0.001 vs vehicle, ##p < 0.01 vs vehicle, and #p < 0.05 vs vehicle. Data is represented as mean ± SEM.

Discussion

Among many pathways that restrict neuroregeneration after an SCI, the RhoA/ROCK pathway serves as a focal point of interest as it is an important regulator for neurite growth.38 In a study conducted by Hara et al.,39 Fasudil, a ROCK inhibitor, was demonstrated to cause more fast and comprehensive neurological recovery in a rat SCI model, compared to methylprednisolone, which had no positive effect on neurological recovery. However, it will not be justifiable to say that the effect of ROCK inhibition arises exclusively from the inhibition of only ROCK, as ROCK acts as a central regulator of several other kinases.40 So, here, we chose LIMK, a particular downstream target of ROCK, and explored its role in the pathology of SCI-associated neurodegeneration.

Myelin-associated inhibitors (MAIs) such as myelin-associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and Nogo transduce a signal after injury to prevent spontaneous axon regrowth.41 The intracellular signaling pathways that combine to alter the actin cytoskeleton, causing growth cone collapse and outgrowth inhibition, are stimulated when MAIs bind to the receptor complex on neuronal growth cones.42 In the adult nervous system, as well as during development, Nogo-A functions as a neuronal local growth suppressor. Its ablation regulates phosphorylation, which, in turn, modifies Rho-GTP and the LIMK1/cofilin pathway. In the intact nervous system, this causes neurite outgrowth, enhanced growth cone motility, and modification of the actin cytoskeleton.43 LIMK1 activation was found to be required for myelin-dependent inhibition since dominant negative LIMK prevented myelin-dependent inhibition in DRG neurons.44 Cofilin, a key regulatory protein of the ADF/cofilin family, controls actin dynamics by severing actin filaments and increasing the off-rate of actin monomers from the pointed actin filament end.45 Increased cofilin phosphorylation is mediated by activated LIMK1, which renders cofilin inactive. Our in vitro results suggested that phosphorylation of LIMK causes a hindrance to microtubule transport and ultimately neurite growth. Inhibition of LIMK by BMS-5 restores growth cone structure, suggesting that, for actin polymerization to be adequately controlled, cofilin cycling is necessary, and hence the inhibition of LIMK could be a promising strategy. Moreover, the inhibition of LIMK by BMS-5 was found to stabilize microtubules by redirecting the growth of tyrosinated tubulin toward the axonal end.

After the initial results of neurite growth obtained from an in vitro study, we checked the expression patterns of phosphorylated LIMKs in vivo through a contusion model of SCI in rats. Hence, we analyzed the expression pattern of both phosphorylated LIMK1 and LIMK2 in a time-dependent manner after SCI in rats. It was found that phosphorylated LIMK1, but not LIMK2, was significantly upregulated at chronic injury time points: DPI-14 and 28. This finding corresponds to the result obtained by Aizawa et al.,46 who used constitutively active LIMK1 to demonstrate that LIMK1 activation was required for Sema3A-induced growth cone collapse and neurite retraction in DRG neurons. Phosphorylated LIMK1, in turn, then inactivated cofilin through its phosphorylation, reducing the amount of cofilin that binds to actin filaments and the extent of breakage of actin filaments. The ratio of nonphosphorylated (active) cofilin to actin filaments is essential for cofilin-mediated actin regulation. Actin filament disintegration happens at low concentrations of nonphosphorylated cofilin, whereas filament stability or nucleation happens at high quantities.47 Our study shows increased p-cofilin in Western blots, suggesting that actin filament disassembly occurs after injury.

As an active (phosphorylated) form of LIMK1 was found to aggravate the neurodegeneration associated with SCI, we decided to target it with the commercially available inhibitor BMS-5. In a previous study by Manaenko et al.,48 BMS-5/LIMKi-3 at a dose of 1 mg/kg reduced brain edema and enhanced neurological function after 72 h in a model of intracerebral hemorrhage (ICH). BMS-5 at this dose reduced ICH-induced LIMK phosphorylation in brain tissues, demonstrating that it can cross the blood–brain barrier (BBB) and affect the CNS. We chose two doses of BMS-5 (0.3 and 1 mg/kg) to inhibit LIMK phosphorylation and to check neuroprotective activity after SCI. As phosphorylated LIMK1 was found to be significantly upregulated at DPI-28, we chose this time point to investigate its effect on each marker. In a model of chronic postsurgical pain, LIMKs were specifically targeted as a therapeutic approach for the management of chronic pain. Here, a single dose (5 μg) intrathecal perioperative administration of BMS-5 significantly and reliably prevented the onset of chronic mechanic allodynia.49 For this particular study, the inhibition of LIMK was found to effectively interfere with the synaptic potentiation process during central sensitization through the regulation of cofilin and cAMP response element-binding protein (CREB). In another study, the effect of ROCK or LIMK inhibition was investigated to check the role of LIMK in cofilin phosphorylation after retinal detachment. According to their findings, cofilin phosphorylation was decreased following retinal detachment when either ROCK or LIMK was inhibited but cofilin expression remained unchanged. The half-life of the ROCK inhibitor, Y27632, was reported to be about 60–90 min in blood, and a similar effect was observed with a significant reduction of p-cofilin in the case of BMS-5 also.50 BMS-5, with a logP value of 3.92, is highly lipophilic and hence assumed to exhibit strong tissue binding after entering the CNS due to hydrophobic interactions. As an amide compound, it has basic characteristics, making it to bind preferentially with negatively charged membranes and tissues.51 Its high logP value suggests that it has a higher binding affinity for efflux transporter, p-glycoprotein (p-gp), enhancing permeability to cross CNS barriers.52

The glucocorticoid drug methylprednisolone, which has been used clinically as an effective therapy for acute traumatic SCI, was found to enhance GAP-43 expression (axon maker) more than the SCI group in an animal model of traumatic SCI with a single bolus dose of 30 mg/kg when administered immediately after injury. According to this finding, methylprednisolone reduced microglia activation, inhibited A1 astrocyte activation, lessened astrocyte cell death, and enhanced functional recovery.53 Another study reported the neuroprotective potential of atorvastatin, administered as a single dose of 5 mg/kg, ip, in a rat compression SCI model. Atorvastatin promoted neurofilaments and GAP-43 positive fiber expressions and markedly enhanced hindlimb motor function after 6 weeks post SCI.54 Both of these studies focused on treating animals during the crucial initial hours following the injury and simultaneously investigated whether administering a therapeutically relevant drug as a single acute dose would successfully reduce the detrimental effects of traumatic SCI. Our results also indicate that a single dose of BMS-5, administered immediately after injury, has been found to be effective through the regulation at the cytoskeletal level. Since LIMK1 was shown to be a BMS-5 target, it was very likely that the neuronal network reorganization was caused by the suppression of cofilin phosphorylation. In the chronic period following SCI, astrocytic, fibrotic, and microglial scars represent significant pathological alterations.55 The present study demonstrates that BMS-5 reduces the extent of scar formation and has a neuroprotective impact. In addition, morphological studies show that the BMS-5-treated group had a smaller lesion area around the epicenter. Finally, compared to the vehicle-treated group, the BMS-5-treated groups had a higher motor function score, indicating a possible role for BMS-5 in enhancing functional recovery. These findings show that the treatment of LIMK inhibitor, BMS-5, may open the way for an alternative strategy toward addressing the impairment associated with SCI. Hence, more research is needed to investigate the novel insights of LIMK inhibitors as a treatment method in individuals with SCI.

Conclusions

Our results showed that (1) phosphorylation of LIMK is associated with microtubule destabilization, and hence, an obstruction in neuronal outgrowth and inhibition of LIMK restores it; (2) increased phosphorylation of LIMK contributes to glial scar formation and neuronal retraction at chronic time points of SCI. As a whole, it can be concluded that LIMK could act as a promising target in SCI-associated neurodegeneration. BMS-5, the LIMK inhibitor, can be an effective compound to promote neuroprotection through the stabilization of microtubules and attenuation in scar formation.

Materials and Methods

Cell Culture and Treatments

Neuro 2a Cell Line from a mouse was purchased from ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum to a final concentration of 10%. The cells were maintained in a 37 °C incubator with 5% CO2. Nocodazole (ab120630, Abcam) was used at a concentration of 30 μM for 30 min as a microtubule inhibitor. Epothilone A (ab143615, Abcam) was used at a concentration of 10 nM for 24 h as a microtubule stabilizing agent. The compound LIM kinase inhibitor, BMS-5, was purchased from Sigma-Aldrich (435930) and used at a final concentration of 5 μM for 24 h as treatment.

Model Induction

Female Sprague-Dawley rats of weights ranging from 220 to 260 g were used to carry out the experimental framework. The animals were procured after they were approved by the Institutional Animal Ethics Committee (IAEC No.: IAEC/2020/036). Guidelines and instructions issued by the IAEC were strictly followed after the procurement of animals. Before performing the surgery, animals were anesthetized using a mixture of ketamine and xylazine with a dose of 75 and 10 mg/kg, respectively, intraperitoneally. An incision was made after determining the T10-T12 vertebrae, following which the laminectomy was performed. Using a spinal cord impactor (68097, RWD), the injury was performed on the T10th level by dropping a tip (weight 10 g, diameter 2.5 mm) from a height of 10 cm. The muscles and skin layers were sutured following surgery. The sham group underwent laminectomy only and was sutured thereafter. Up to the first week following surgery, gentamicin (i.m.) and buprenorphine (i.p.) were given twice a day. Until a normal urine function was restored, the urinary bladder was emptied twice daily.

Administration of the Compound

The compound LIM kinase inhibitor BMS-5/LIMKi-3 was purchased from Sigma-Aldrich (435930). It was administered intraperitoneally, 1 h post injury. The stock solution was prepared in DMSO. It was administered with two different single doses of 0.3 and 1 mg/kg.

Immunocytochemistry

Approx. 50,000 cells were seeded in DMEM with FBS on a coverslip. After 24 h, treatment media was given and further incubated for another 24 h. Then, the media was removed and washed with PBS. After washing, cells were fixed in 4% PFA for 15 min at 37 °C. Next, the cells were washed with PBS three times. After that, cells were permeabilized with 0.5% Triton-X in 1× PBS for 10 min. Cells were quickly rinsed thereafter with 1× PBS. Then, a protein blocker (ab64226, Abcam) was used for 15 min for blocking. After that, cells were incubated with primary antibodies: antitubulin antibody, detyrosinated (1:250, AB3201, Sigma-Aldrich) and antitubulin antibody, tyrosinated (1:250, MAB1864-I, Sigma-Aldrich) at 4 °C overnight. Next, cells were washed with PBS 3 times. After that, the mixture was incubated with secondary antibodies: donkey anti-rabbit Alexa Fluor 488 (1:1000, ab150073, Abcam) and goat anti-rat Alexa Fluor 555 (1:1000, A-21434, Thermo Fisher Scientific) for 1 h in the dark. Then, cells were washed 3 times with PBS for 10 min each. Phalloidin Atto 520 (54367, Sigma-Aldrich) was used for 10 min at a dilution of 1:100 to stain F-actin. Then, after 3 washings with PBS, DAPI (D9542, Sigma-Aldrich) was used to stain the nuclei. Finally, cells were mounted with DPX. The quantification for tyrosinated tubulin was measured for each cell as corrected total cell fluorescence (CTCF) = integrated density -(area of selected cell × mean fluorescence of background readings), using ImageJ.

Western Blot Analysis

Tissue from the epicenter region of the spinal cord, having a length of approximately 2 cm and weighing approximately 70–90 mg, was taken for Western blot analysis. An equal amount of protein (30 μg) samples was loaded in each well and was separated by SDS PAGE. The protein samples were then transferred to a PVDF membrane through TransBlot assembly (Bio-Rad). Then, blocking was done with 5% BSA for 1 h at room temperature. After that, the membranes were incubated with primary antibodies at 4 °C overnight: p-LIMK1 (1:750, ab194798, Abcam), p-LIMK2 (1:1000, SAB4504461, Sigma-Aldrich), p-cofilin (1:1000, 44–1072G, Invitrogen), LIMK1 (1:1000, PA5–14938, Thermo Fisher Scientific), cofilin (1:1000, PA5–27627, Thermo Fisher Scientific), GFAP (1:10000, 13–0300, Invitrogen), β-tubulin III (1:1000, T2200, Sigma-Aldrich), occludin (1:1000, 33–1500, Thermo Fisher Scientific), KIF5B (1:5000, ab167429, Abcam), and GAPDH (1:10000, ab8245, Abcam). Followed by incubation, the membranes were washed three times with TBS Tween (TBS-T). Then, they were incubated with HRP-conjugated secondary antibody (1:10000, ab6721, Abcam) (1:10000, ab205719, Abcam) for 1 h. After that, the membranes were washed again with TBS-T and visualized using an enhanced chemiluminescence solution (Bio-Rad; 170–5061) in GelDoc (Bio-Rad). The densitometric analysis was performed by using ImageJ software. For quantification purposes, the mean gray value for each marker and their corresponding loading controls was measured. After the background measurement was subtracted, the final relative quantification values were obtained as the ratios of the net band to net loading control.

Quantitative Real-Time PCR

Animals were sacrificed at DPI-28, and the injury epicenter region of the spinal cord, having a length of approximately 2 cm and weighing approximately 70–90 mg, was isolated. The RNAs were extracted using TRI Reagent (T9424, Sigma) according to the manufacturer’s protocol and quantified using the Nanodrop method (Thermo Scientific, NanoDrop 2000C). From RNA, cDNA was synthesized using a cDNA synthesis kit (1708891, Bio-Rad), using a Thermocycler. Diluted cDNA samples and specific primers were used in a qRT-PCR machine (Bio-Rad) to conduct PCR following the manufacturer’s instructions. The reaction was performed with the following parameters: an initial step at 95 °C for 10 min, a second phase at 95 °C for 15 s, and an annealing temperature of 60 °C for 30 s for 40 cycles accompanied by melt curve analysis. Using 18S as an internal control, the data was represented as the Ct or cycle threshold values.

Tissue Fixation and Paraffin Block Preparation

The animals were anesthetized and transcardially perfused with 0.9% saline and 4% PFA. After the spinal cord was removed, it was immersed in 4% PFA at 4 °C overnight. For 2 h each, the spinal cord was dehydrated gradually in a gradient of alcohol (70, 80, 90, and 100%) and then in a mixture of xylene and alcohol (1:1). A paraffin embedder machine (Thermo Fisher Scientific) was used to make the paraffin blocks of the spinal cord. The blocks were cut longitudinally, and 5 μm sections were taken using a microtome (Leica).

Immunohistochemistry

Tissue sections were rehydrated in a series of gradient alcohols, and then pepsin (R2283, Sigma-Aldrich) was used for antigen retrieval. This was followed by adding 3% hydrogen peroxide, which was used to inhibit endogenous peroxidase activity. After a quick rinse in PBS, the nonspecific interactions in the sections were blocked with a protein blocker (ab64226, Abcam) and then incubated with primary antibodies at 4 °C overnight. The primary antibodies used were p-LIMK1 (1:100, ab194798, Abcam), laminin (1:75, L9393, Sigma-Aldrich), GFAP (1:500, ab4674, Abcam), β-tubulin III (1:500, T2200, Sigma-Aldrich), and IBA1 (1:100, PA5–18039, Thermo Fisher Scientific). Secondary antibodies used were Alexa Fluor 647 (1:1000, ab150079, Abcam), Alexa Fluor 555 (1:1000, ab150134, Abcam), and Alexa Fluor 568 (1:1000, ab175477, Abcam). After a series of PBS-T washes, Alexa Fluor-conjugated secondary antibodies were incubated for 1 h at room temperature, away from light. Each section was then stained for 10 min with DAPI (1 μg/mL) to stain the nuclei. The sections were mounted with DPX after they were rinsed twice more with PBS-T. A fluorescence image was obtained by examining slides with confocal microscopy (Leica) at the required laser wavelength. The fluorescence intensity was analyzed by using ImageJ software. For this purpose, the RGB measure was selected from the ImageJ plugin. Then, from the measured window, the mean value was selected for individual markers and plotted in the GraphPad Prism.

Functional Recovery

Using the Basso–Beattie–Bresnahan (BBB) scoring method, the hindlimb locomotor functional recovery was assessed on DPI-1, 7, 14, 21, and 28 in sham, vehicle, and BMS-5-treated animals in accordance with the scoring system. Animals were placed in an open-field area and scored using predefined criteria. The scale runs from 0 to 21, with 0 indicating no observed hindlimb movement and 21 indicating complete functional recovery.56

Quantification of Lesion Size

Lesion size was quantified in ImageJ (NIH) using the longitudinal sections of the spinal cord with GFAP and collagen IV immunoreactivity. The lesion area was found by outlining the scar in the epicenter region of the spinal cord in ImageJ with a freehand drawing tool and then automatically measured, and the value was recorded.

Statistical Analysis

GraphPad Prism (GraphPad Software 8.3.0) was used for the statistical analysis. Using Student’s t test, two groups were compared. Multiple groups were compared using 1-way ANOVA. For multiple groups with two independent variables, 2-way ANOVA was conducted. p-Value <0.05 was considered statistically significant. For all experiments for each time point, 3 individuals were used to confirm the validity and reproducibility of any observed changes. However, other than using 3 biological replicates, in some experiments like immunohistochemistry, technical replicates were also used to characterize the precision and variability of the method.

Acknowledgments

This work was supported by the Department of Science and Technology grant, Science and Engineering Research Board (DST-SERB) (SRG/2020/000706), and NIPER Ahmedabad. Abhishek Roy received a fellowship from the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India. Zarna Pathak received a fellowship from the Indian Council of Medical Research (SRF_2021-11932). The authors would like to thank the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Govt. of India, and NIPER Ahmedabad. The paid version of Biorender.com was utilized to prepare the illustrations.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.3c00272.

  • Hematoxylin and eosin staining for longitudinal spinal cord sections in sham and DPI-28 groups (PDF)

Author Contributions

A.R.: methodology, molecular biology studies, statistical analysis, and writing the original draft of the manuscript. Z.P.: editing of the manuscript. H.K.: conceptualization, confocal microscopy, supervision, reviewing, and finalizing of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt3c00272_si_001.pdf (165.7KB, pdf)

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