PAK4 suppresses motor neuron degeneration in hSOD1G93A‐linked amyotrophic lateral sclerosis cell and rat models

Abstract Objectives Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons (MN). CREB pathway‐mediated inhibition of apoptosis contributes to neuron protection, and PAK4 activates CREB signalling in diverse cell types. This study aimed to investigate PAK4’s effect and mechanism of action in ALS. Methods We analysed RNA levels by qRT‐PCR, protein levels by immunofluorescence and Western blotting, and apoptosis by flow cytometry and TUNEL staining. Cell transfection was performed for in vitro experiment. Mice were injected intraspinally to evaluate PAK4 function in vivo experiment. Rotarod test was performed to measure motor function. Results The expression and activation of PAK4 significantly decreased in the cell and mouse models of ALS as the disease progressed, which was caused by the negative regulation of miR‐9‐5p. Silencing of PAK4 increased the apoptosis of MN by inhibiting CREB‐mediated neuroprotection, whereas overexpression of PAK4 protected MN from hSOD1G93A‐induced degeneration by activating CREB signalling. The neuroprotective effect of PAK4 was markedly inhibited by CREB inhibitor. In ALS models, the PAK4/CREB pathway was inhibited, and cell apoptosis increased. In vivo experiments revealed that PAK4 overexpression in the spinal neurons of hSOD1G93A mice suppressed MN degeneration, prolonged survival and promoted the CREB pathway. Conclusions PAK4 protects MN from degeneration by activating the anti‐apoptotic effects of CREB signalling, suggesting it may be a therapeutic target in ALS.

widely used in the research of ALS aetiology and treatment. 11 The exact mechanism underlying MN degeneration in ALS is still mostly unknown. Currently, only riluzole and edaravone are approved by FDA for ALS clinical therapy. However, they could provide limited efficacy. 12,13 Thus, strategies to promote MN survival remain an urgent need for ALS treatment.
cAMP-response-element binding protein (CREB) is a crucial transcription factor in the brain that regulates essential physiological functions including neuronal excitability, dendritic growth, long-term synaptic plasticity formation and neuronal survival. [14][15][16][17][18][19][20] CREB mediates neuroprotection mainly via its downstream genes, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a), 21 brain-derived neurotrophic factor 22 and B-cell lymphoma 2 (Bcl-2). 23 Deregulation of CREB signalling is associated with several neurodegenerative diseases, for instance, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and ALS. 22,[24][25][26] Restoring CREB activation rescues defects in dendrite morphogenesis in TDP-43 misregulated neurons. 27 In particular, human adipose stem cell extract treatment improves MN function and prolongs the life span in ALS mice by increasing the expression of pCREB. 28 Moreover, our group has demonstrated that the neuroprotective effect of lithium for ALS relies on attenuating the improper binding of mutant SOD1 protein aggregates with pCREB. 29 Although CREB signal transduction networks are complex and obscure, it has been proved that the CREB pathway plays a neuroprotective role in ALS disease.
The serine/threonine p21-activated kinases (PAKs) are comprised of six mammalian proteins that are divided into group I (PAK1 to PAK3) and group II (PAK4 to PAK6) in terms of their structural and functional features. 30 Group II PAKs contribute to a wide range of intracellular processes including cytoskeletal dynamics, cell growth, tumorigenesis, neuronal dysfunction and cell survival. [30][31][32][33][34] Studies on PAK4 have revealed its pivotal role in cytoskeletal remodelling, neuronal development, axonal outgrowth and neuronal survival. 24,35,36 PAK4 knockout mice display dramatic defects in the heart and nervous system, with striking abnormalities observed in axonal outgrowth and neural tube formation. 36 Studies have shown that PAK4 expression in PD patients is downregulated and that overexpressing it promotes DA neurons survival in the a-synuclein rat model. 24 Deregulation of AKT signalling is involved in ALS. Restoring AKT activation rescues MN from death in ALS. [37][38][39][40] PAK4 is linked to AKT activity 41,42 ; thus, PAK4 may promote the survival of MN through AKT. Together, these studies suggest that PAK4 plays a pivotal role in neuronal pathophysiology.
PAK4 has been shown to increase the levels and activation of CREB globally in diverse cell types. 24,[43][44][45] Given that PAK4 targets CREB and that CREB promotes MN survival, maintaining PAK4 activation is expected to play a pivotal role in preventing the progressive loss of MN in ALS. Therefore, we hypothesized that decreased PAK4 activation might involve in the pathogenesis of ALS. In this study, we found that the expression and activation of PAK4 were deregulated in ALS models. Overexpression of PAK4 protected MN from hSOD1 G93A -induced apoptosis, and this neuroprotective effect was alleviated by CREB inhibitor. Finally, hSOD1 G93A mice injected with LV-PAK4 manifested a delay in disease onset, prolongation of survival and promotion of CREB signalling. These data suggest that the neuroprotective role of PAK4 in ALS MN is mediated by the CREB pathway.

| Animals
Transgenic mice carrying the hSOD1 G93A gene on the C57BL/6J strain background were obtained from Jackson Laboratory (Bar Harbor, ME, USA). All mice were bred and maintained on a 12 hours day/night cycle with adequate food and water. The offspring were identified by polymerase chain reaction (PCR) analysis of tail DNA. 46 Transgenic female mice were culled at postnatal day 75, 120 and 140, corresponding to presymptomatic, early-sym stage and latesym stage of the disease. Age-matched non-transgenic female littermates were used as wild-type controls. All animal experiments were performed in accordance with the International Animal Care Guidelines and were approved by the Experimental Animal Ethics Committee of Harbin Medical University.

| Generation of a hSOD1G93A-transfected NSC34 cell line and cell cultures
The mouse neuroblastoma × spinal cord NSC34 cell line was a gift from Cedarlane Laboratories (Vancouver, Canada). NSC34 cells were stably transfected with hSOD1 G93A (mSOD1), human wild-type SOD1 (wtSOD1) or an empty lentivirus vector (pLV) separately as described. 47 Puromycin at 200 μg/mL (G418, Invitrogen) was used to maintain a stable cell line translation.
The spinal cords were dissected and fixed in 4% PFA for 24 hours, and then were embedded in paraffin. 3 μm sections of cervical or lumbar enlargements were prepared for immunohistochemistry and immunofluorescence staining. In brief, after deparaffinization and dehydration, sections were incubated with a primary antibody against PAK4 (1:200, Santa Cruz Biotechnology) overnight at 4°C.
For the visualization, sections were incubated with secondary antibodies followed by DAB (3,3'-diaminobenzidine) staining. The images were captured using a Leica microscope (Leica, Wetzlar, Germany) and analysed by Image-Pro Plus 6.0 software (Media

| Western blotting analysis
The whole spinal cords from mice or prepared cells were lysed in RIPA lysis buffer (Beyotime Biotechnology Co., Ltd.) containing phosphatase and protease inhibitor mixture (Roche, 4906845001 and 04693132001). Lysates were centrifuged at 18630 g for 20 minutes at 4°C, and only the supernatants were analysed by Western blotting, as described previously. 47 Primary antibodies: rabbit anti-PAK4 (1:1000, Cell Signaling Technology), rabbit anti-pPAK4 at Ser474

| qRT-PCR
For PAK4 mRNA quantification, total RNA was extracted from spinal cords or prepared cells using Trizol (Cwbio); cDNA was generated using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO CO., LTD. Life Science Department OSAKA JAPAN).
For miR-9-5p detection, total RNA was reverse-transcribed using The relative expression of PAK4 mRNA and miR-9-5p was analysed using the 2 −ΔΔCT method. qRT-PCR reactions were carried out in three independent runs in triplicate.
The effectiveness of transfection was evaluated by qRT-PCR and Western blotting.

| Plasmid transfection and treatment
The cDNA plasmid (OriGene, Beijing, China) mediated overexpression of PAK4 in mSOD1 cells was performed as previous description 48

| Cell apoptosis assay
To detect apoptosis, at 72 hours after siPAK4 transfection or 3i

| Disease course analysis and behaviour tests
To test motor function, rotarod performance was examined in LV-PAK4 or LV-cherry injected hSOD1 G93A mice starting at 80 days of age. After 1 week of training, each mouse was placed on the rotating rod at 16 rpm every 2 days to record the latency time before the mouse would fall off. The test was repeated three times for each animal. Disease onset was defined as the age at which a mouse could no longer stay on the rotating rod for 180 seconds. The date of disease endpoint was recorded when the mouse could not rise within 30 seconds after being placed on its side.

| Statistical analyses
The data were analysed using SPSS version 23.0 and GraphPad Prism 8.0 software (GraphPad Software, USA). Statistical significance was evaluated with Student's t-test or one-way analysis of variance (ANOVA). Quantitative data were expressed as mean values of at least three determinations ± SD. Tests were considered statistically significant at P < .05.

| Reduced PAK4 expression in the spinal cord of hSOD1G93A mice
Immunofluorescence analyses of the spinal cords of hSOD1 G93A mice and non-transgenic littermates (WT) were performed to detect differences in PAK4 levels associated with ALS. Results showed that for hSOD1 G93A mice, the number of MN (green) was significantly reduced, and MN exhibited a weaker fluoresence of PAK4 (red) compared with those of WT ( Figure 1A). Decreased PAK4 expression in ALS was confirmed by Western blotting. We also found that the protein levels of phospho-PAK4 s474 , an index of PAK4 activation, were lower in p140 hSOD1 G93A mice compared to WT ( Figure 1B and C). The pPAK4/PAK4 ratio was greater in hSOD1 G93A mice ( Figure 1D). These data suggested that the expression and activation of PAK4 in hSOD1 G93A mice were both downregulated. Next, we analysed PAK4 mRNA levels in the spinal cords of hSOD1 G93A mice (n = 6) and WT (n = 6) using qRT-PCR. It was found that the gene-expression pattern was followed by protein expression. The levels of PAK4 mRNA were significantly reduced in hSOD1 G93A mice ( Figure 1E).
To characterize the temporal expression patterns of PAK4 in ALS mice, we evaluated PAK4 levels in hSOD1 G93A and WT mice at three stages of the disease (the presymptomatic stage P75, early-sym stage P120 and late-sym stage P140) using immunohistochemistry. Results showed that compared with WT, PAK4 immunoreactivity was significantly lower at early-sym and latesym stages of the disease in ALS mice (Figure 2A and Figure S1A).
However, at the presymptomatic stage, there was no statistical difference in the optical density (OD) values of PAK4 between hSOD1 G93A mice and WT ( Figure 2B and Figure S1B). The levels of PAK4 at three stages of the disease were further confirmed by Western blotting ( Figure 1B and Figure S2). Taking these data together, we predicted that PAK4 was involved in the pathophysiological process of ALS.

| PAK4 protein levels are downregulated in mSOD1 cells
Next, we explored whether the expression and activation of PAK4 were altered in vitro models of ALS. NSC34 cells exhibit many MN characteristics, including the generation of action potentials, expression of neurofilament proteins and synthesis of acetylcholine. [49][50][51] Our group has confirmed that choline acetyltransferase, an MN marker, is expressed in NSC34 cells. 40 Thus, NSC34 cells stably transfected with hSOD1 G93A (mSOD1) were used as a cell model of ALS in subsequent studies. Immunocytochemistry staining showed that compared to pLV cells, mSOD1 cells displayed weaker fluorescence intensity of PAK4, while wtSOD1 cells displayed a similar intensity ( Figure 3A). Decreased PAK4 expression in mSOD1 cells was confirmed by Western blotting. We also found that compared with pLV, protein levels of pPAK4 were significantly decreased in mSOD1 cells, while wtSOD1 and pLV cells had similar pPAK4 protein levels ( Figure 3B and C). The pPAK4/PAK4 ratio in mSOD1 cells was greater than in control pLV and wtSOD1 cells ( Figure 3C). These data suggested that the expression and activation of PAK4 in mSOD1 were both downregulated. Next, qRT-PCR analysis suggested that PAK4 mRNA levels of mSOD1 cells were robustly downregulated to approximately 18.9% compared to pLV cells. PAK4 mRNA expression in wtSOD1 and pLV cells showed no statistical difference ( Figure 3D).

| Knockdown of PAK4 promotes motor neuron degeneration through inhibiting CREB pathway
To determine the biological role of PAK4 in MN, we selected NSC34 cells with many MN characteristics for further study. Three siR-NAs targeting PAK4 were transfected into NSC34 cells, and the efficiency of PAK4 knockdown was evaluated by Western blotting and qRT-PCR ( Figure 4A). We selected siPAK4-2 for subsequent F I G U R E 1 Decreased PAK4 expression and activation in motor neuron of hSOD1 G93A mice. A, Confocal microscopy images showed that PAK4 expression in MN of p140 hSOD1 G93A mice was lower than that of non-transgenic littermates (WT), n = 3/group. The number of MN surviving in hSOD1 G93A mice was significantly decreased compared with WT (cervical spinal cord: 41.00 ± 3.61 vs 13.67 ± 2.08; lumbar spinal cord: 44.33 ± 4.04 vs. 13.33 ± 3.05; P < .05). B, Immunoblotting of spinal cord extract from p140 hSOD1 G93A mice and WT was conducted to detect PAK4 and pPAK4 expression. C, Quantification of PAK4 and pPAK4 blots in (B) normalized to β-actin. Relative PAK4 and pPAK4 protein levels in hSOD1 G93A mice were significantly downregulated. D, pPAK4/PAK4 ratio in spinal cords of hSOD1 G93A mice was high. E, qRT-PCR revealed that hSOD1 G93A mice showed remarkably decreased PAK4 mRNA levels (n = 6/group). Data were shown as means ± SD. Student's t-test was used to evaluate statistical significance, *P < .05, **P < .01, ***P < .001 experiments because of its obvious knockdown effects. Flow cytometry analysis suggested that NSC34 cells transfected with siPAK4-2 yielded a significantly higher rate of cell apoptotic death (49.34 ± 0.95%) than siNC control (32.07 ± 2.45%; Figure 4B and C). Consistently, TUNEL staining indicated that knockdown of PAK4 significantly increased the percentage of cells experiencing apoptotic death (35.67 ± 2.51%), and in the siNC group, the percentage of cell apoptosis was 20.57 ± 1.50% ( Figure 4D and E). These data suggested that PAK4 protected NSC34 cells from apoptotic death.
Next, we further investigated the mechanism by which PAK4 promoted cell survival. Previous studies have revealed that PAK4 is a critical regulator of CREB and that CREB promotes MN survival. 52 Thus, Western blotting of PAK4, CREB, pCREB and the CREB target proteins (PGC-1a and Bcl-2) was performed with extract from siNC and siPAK4-2 groups. The results showed that the levels of CREB, pCREB, Bcl-2 and PGC-1a were decreased in PAK4 knockdown NSC34 cells. These data indicated that PAK4 might inhibit MN degeneration via the CREB pathway ( Figure 4F and G).

F I G U R E 2
The expression of PAK4 decreased in the cervical spinal cords of hSOD1 G93A mice during disease progression. Paraffin sections of cervical spinal cords from hSOD1 G93A mice and WT at three stages of the disease were subjected to immunohistochemistry. A, PAK4-stained MN (maximum diameter ≥ 20 μm) were detected in the anterior horn of spinal cords from both hSOD1 G93A mice and WT. Scale bar = 50 μm. B, At stages p120 and p140, OD values of PAK4 significantly decreased in hSOD1 G93A mice, while there was no deregulation at stage p75 (n = 3/group, six sections/mouse). Data were provided as means ± SD and were tested for significance using Student's t-test. ns ≥ .05, *P < .05

| CREB pathway mediates PAK4-induced neuroprotection in mSOD1 cells
To investigate whether the neuroprotective role of PAK4 would extend to cell models of ALS, we upregulated PAK4 expression using a Thus, these data suggested that CREB and its target proteins BCL-2, PGC-1a mediated the neuroprotective effect of PAK4.

F I G U R E 3
Reduced expression of PAK4 is identified in mSOD1 cells. A, Confocal microscopy images of PAK4 (red) in pLV, wtSOD1 and mSOD1 cells, respectively. The weakest intensity of PAK4 staining was observed in mSOD1 cells, while similar intensities were seen in wtSOD1 and pLV cells. Scale bar = 50 μm. B, Immunoblotting analysis of PAK4 and pPAK4 protein expression levels was conducted with lysates from control pLV, wtSOD1 and mSOD1 cells. C, Quantification of PAK4 and pPAK4 blots in (B) normalized to β-actin. Relative PAK4 and pPAK4 protein levels were significantly decreased in mSOD1 cells. pPAK4/PAK4 ratio was highest in mSOD1 cells. D, qRT-PCR analysis detected the mRNA expression of PAK4. For relative quantification of PAK4 mRNA expression, the 2 −ΔΔCt method was conducted using β-actin for normalization; data were provided as means ± SD. ANOVA with Student-Newman-Keuls post hoc analysis was used to evaluate statistical significance, ns ≥ .05, *P < .05, **P < .01, ***P < .001

| PAK4 deficiency increases motor neuron degeneration by inhibiting CREB pathway in mSOD1 cells and hSOD1G93A mice
To explore the effects of deregulated PAK4 expression on downstream consequences and neurodegeneration in ALS models, the protein levels of CREB signalling and cleaved-caspase3 were analysed in mSOD1 cells and the spinal cords from hSOD1 G93A mice.
Immunoblotting analysis revealed that mSOD1 cells exhibited low levels of CREB, pCREB and CREB target proteins than pLV cells, while wtSOD1 cells displayed similar levels ( Figure 7A and B). higher levels of cleaved-caspase3 than control ( Figure 7D and F).
These data suggested that downregulated PAK4 expression promoted neurodegeneration in ALS models by impairing the CREB pathway.

| PAK4 is negatively regulated by miR-9-5p in ALS
MicroRNAs (miRNAs), small non-coding RNAs, repress gene translation and promote target mRNA degradation. 53 It has been reported that PAK4 is target regulated by a variety of miRNAs, including miR-433, 54 miR-224, 55 miR-199a-3p 56 and miR-9-5p. 57 Moreover, the expression of miR-9-5p in the cerebrospinal fluid of ALS patients is increased. 58 We speculated that PAK4 might be inversely regulated by miR-9-5p in ALS. To test our hypothesis, we first analysed miR-9-5p levels in ALS vivo and vitro models using qRT-PCR. Results showed that compared with control, the expression of miR-9-5p in spinal cords of p140 hSOD1 G93A mice was increased, especially in the mSOD1 cell line ( Figure 8A). In studies, we have found that the mRNA levels of PAK4 were decreased in vivo and in vitro models of ALS ( Figures 1E and 3D). To determine whether miR-9-5p negatively regulated PAK4 mRNA levels, miR-9-5p inhibitor transfection was conducted on mSOD1 cells. The results showed that downregulation of miR-9-5p significantly upregulated the expression of PAK4 mRNA and protein ( Figure 8B).

| Delayed disease onset and extended lifespan of hSOD1G93A mice injected with LV-PAK4
Since PAK4 played a neuroprotective role in vitro, we next evaluated the in vivo biological function of PAK4 in hSOD1 G93A mice.
We injected hSOD1 G93A mice intraspinally at 60 days of age with

| Overexpression of PAK4 protects motor neurons from degeneration through enhancing CREB signalling in hSOD1G93A mice
To test the effects of PAK4 overexpression on MN degeneration, we performed immunostaining with anti-MAP-2 to examine the number of MN in the spinal cords of LV-PAK4 treated hSOD1 G93A mice.
We found that the number of MN surviving in LV-PAK4-injected hSOD1 G93A mice was significantly increased compared with LVcherry-injected hSOD1 G93A mice (cervical spinal cord: 32.00 ± 3.61

F I G U R E 8
The expression of miR-9-5p increases in ALS and reversely regulates PAK4 mRNA levels. A, The expression levels of miR-9-5p in spinal cords from hSOD1 G93A mice and in mSOD1 cells were determined by qRT-PCR. B, The expression of miR-9-5p and PAK4 at mRNA and protein levels in mSOD1 cells transfected with anti-miR-9-5p or miR-NC were determined by qRT-PCR and Western blotting. Data were presented as means ± SD. *P < .05, **P < .01, ***P < .001. Statistical analyses were performed using Student's t-test Data represent means ± SD. Statistical analyses were performed using Student's t-test or Kaplan-Meier survival analysis. *P < .05, **P < .01, ***P < .001 mice ( Figure 10C and E). These data suggested that PAK4 exerted neuroprotective effects by activating CREB signalling in the hSOD1 G93A mouse models of ALS.

| D ISCUSS I ON
Our results reveal that PAK4 is a potential effector of MN degeneration in ALS. We found that the expression and activation of PAK4 were downregulated in vivo and in vitro models of ALS.
Overexpression of PAK4 protected MN from hSOD1 G93A -induced apoptosis. Notably, introducing LV-PAK4 in hSOD1 G93A mice suppressed MN degeneration and thereby substantially extended mouse lifespan. Mechanistic studies revealed that CREB pathway in mSOD1 cells and hSOD1 G93A mice was inhibited and that the alteration was suppressed by overexpression of PAK4. We have also found that 3i, which inhibits CREB, compromised the ability of PAK4 to relieve mSOD1-induced cytotoxicity. Taken together, our data suggested that PAK4 exerted neuroprotective effects for ALS MN by activating the CREB signalling.
PAK4 expresses ubiquitously and plays an essential role in multiple biological processes. 36,59 Dysregulation of PAK4 expression involves in various diseases. 60 The functions of PAK4 in diseases have principally been investigated in cancers, and overexpression of it contributes to tumorigenesis. [61][62][63] In the nervous system, PAK4 is required for neuronal development and axonal outgrowth. 35,36 In dopaminergic neurons of PD patients, the expression and activation of PAK4 are downregulated and this decline may promote PD pathogenesis. 24 However, the relationship between PAK4 and ALS disease remains unclear. In this context, we demonstrate for the first time that PAK4 protein expression as well as activation significantly declined in cells and mice carrying hSOD1 G93A .
Considering that the expression of PAK4 declined at early-sym stages, PAK4 might be involved in ALS disease progression.
A series of findings have shown that activated CREB promotes neuronal survival through a transcription-dependent pathway. [14][15][16][17]20 Moreover, the activation of CREB-mediated downstream proteins plays a neuroprotective role in PD and AD rat models. 22,24,64 Increasing expression of pCREB can improve MN function and prolong the life span of ALS mice. 28 In accordance with previous studies, we found that PAK4 protected MN from mSOD1-induced cytotoxicity through increasing CREB expression and activation. 3i ameliorated the neuroprotective effects of PAK4 and increased apoptosis by inhibiting the transcription activity and phosphorylation of CREB in ALS cell models. Thus, we further confirmed the protective role of CREB in the normal nervous system and neurodegenerative diseases.
Accumulating evidence suggests that CREB is a target of PAK4.
In the present study, we found that knockdown of PAK4 reduced   24,75 Moreover, PAK signalling is stimulated by ALS2; mutations in the ALS2 gene cause some rare juvenile forms of ALS. 76,77 Our findings demonstrated that PAK4 plays a neuroprotective role in ALS models. In aggregate, these studies suggest that dysregulation of PAKs is a pathogenic mechanism in a series of neurological diseases. Thus, the development of more selective and effective interventions targeting PAKs may be a promising therapeutic strategy.
Many studies involving ALS are performed in one gender. 78

| CON CLUS IONS
This work demonstrates that PAK4 protein levels and activation are downregulated in hSOD1 G93A -linked ALS cell and rat models, and overexpression of it protects MN from degeneration.
The neuroprotective effect of PAK4 is mediated by activation of CREB-mediated neuroprotective signalling pathway. miR-9-5p is responsible for the decreased expression of PAK4 in mSOD1cells.
Our data show that PAK4 may be a potential therapeutic target in ALS.A

ACK N OWLED G EM ENTS
We are very grateful to Dr Shengwang Liu from the Harbin Veterinary University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used during the current study are available from the corresponding author on reasonable request.