ALS is a relentless and fatal neurodegenerative disease. Development of neuroprotective treatments to prevent or delay the progression of the disease remains an unachieved goal. Here, we validate for the first time that PAK4 is a crucial survival factor for MN. The expression and activity of PAK4 are downregulated in vivo and in vitro models of ALS. Activation or overexpression of PAK4 and its downstream signaling pathways might serve as a new potential therapeutic method for ALS.
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 [61–63]. In the nervous system, PAK4 is required for neuronal development and axonal outgrowth [35, 36]. In dopaminergic neurons of PD patients, the expression and activity of PAK4 are down-regulated 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 activity significantly declined in cells and mice carrying hSOD1G93A. 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 [14–17, 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 p-CREB 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 activity. 3i amelior- ated 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 the levels and activity of CREB in NSC34 cells. Overexpression of PAK4 increased CREB levels and activity in mSOD1 cells. In mice and cell models of ALS, the low expression of PAK4 caused inhibition of the CREB pathway and promoted MN degeneration. Cumulatively, our findings are consistent with previous works that PAK4 sustains high CREB levels and improves CREB activity in various tissues [43, 44]. However, it is unclear how PAK4 maintains high levels of CREB. Additional studies are required to further elucidate the relationship between PAK4 and CREB expression and degradation. PAK4 could not directly phosphorylate CREB in vitro, suggesting that PAK4 activated CREB transcription by maintaining a high level. Whether PAK4 increases the transcriptional activity of CREB through a phosphorylation-independent pathway in ALS needs subsequent study.
Growing evidence reveals that mitochondrial dysfunction plays a crucial role in ALS [65]. PAK4 can inactivate the pro-apoptotic protein BAD (members of the Bcl-2 family pro- apoptotic activities) by specifically phosphorylating S112 [66]. This phosphorylation protects cells from apoptosis by inhibiting BAD localization in the mitochondrial outer membrane. siRNA treatment decreased PAK4 expression in parallel with inhibition of the CREB downstream effector proteins PGC-1a and Bcl-2. Conversely, overexpression of PAK4 promoted the levels of Bcl-2 and PGC-1a in cell models of ALS. Moreover, 3i decreased PAK4-induced expression of the CREB downstream proteins Bcl-2 and PGC-1a, thereby reducing cell survival. These data demonstrate that the up-regulation of Bcl-2 and PGC-1a mediates PAK4-induced neuroprotection. Considering the close association of PGC-1a and Bcl-2 with mitochondrial health [67, 68], PAK4 may contribute to mitochondrial protection in a transcription-dependent manner. Thus, transcription-dependent and transcription- independent theories could explain the mechanisms that PAK4 protects mitochondria in the presence of neurotoxic stimuli.
Although PAK4 is a potential therapeutic target for ALS, persistent expression of it in the spinal cord after gene therapy might contribute to tumorigenesis because PAK4 is a potent oncogene. However, we did not monitor tumor formation in LV-PAK4 injected hSOD1G93A mice, but we did not maintain lengthy follow-up. In addition, a lower cancer risk was observed in human ALS patients after diagnosis compared with healthy individuals [69]. Thus, we speculate that a lack of tumorigenesis is likely due to a poor microenvironment for cell survival and proliferation in neurodegenerative diseases. ALS is characterized by progressive death of upper and lower MN, suggesting that the brain and spinal cord could not provide a proliferative environment. Moreover, most primary brain tumors develop from glial and meningeal cells. Thus, although PAK4 is regarded as an oncogenic gene, if a local delivery method can guarantee MN-specific targeting, this could prevent undesirable effects and provide a promising therapeutic intervention for ALS. We also consider CREB as another target for modulation of the PAK4 neuroprotective pathway. CREB may be less potent than PAK4 because the role of CREB is more restricted compared with PAK4, but it may specifically enhance the expression of Bcl-2 with a reduced risk of tumor formation.
Group I of the PAK proteins plays a vital role in spinocerebellar ataxia type 1 (SCA1) [52], fragile X syndrome (FXS) [70], X-linked mental retardation [71], and neurodegenerative diseases such as HD [31, 32] and AD [72]. Rescue from disease pathology in cell and mice models of SCA1 and improvement of abnormal behavior in FXS mice model by group I PAK inhibitors, offer proof of PAKs as therapeutic targets [73, 74]. Recent studies have implicated PAK4 and PAK6 in PD by demonstrating that PAK4 plays a neuroprotective role in PD models and that PAK6 is a downstream regulator of PD-causing mutations [24, 75]. Moreover, PAK signaling 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.
In summary, the current findings identify a neuroprotective role for PAK4 in ALS models. A limitation of this study is the absence of human postmortem brain samples; accordingly, we did not study how PAK4 expression levels correlate with ALS in human patients. Further work needs to address the mechanism for PAK4 increasing the levels of CREB and determine if there are other mechanisms for PAK4 enhancing CREB transcriptional activity in ALS. The neuroprotective pathway of PAK4 still needs to be better clarified before targeting PAK4 as a therapeutic strategy in patients with ALS.