p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1.

Aggregation of misfolded alpha-synuclein (α-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying α-synuclein aggregation has been intensively studied in Parkinson's disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of α-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and α-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4S445N/S474E), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced α-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4DAT-CreER mice compared to controls. Conversely, caPAK4 reduced α-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of α-synuclein in the AAV-α-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of α-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.

Hounsfield unit measurement method and related factors that most appropriately reflect bone mineral density on cervical spine computed tomography.

OBJECTIVE: Our study's purpose was to determine the most reliable Hounsfield unit (HU) measurement method to reflect bone mineral density (BMD) on cervical spine computed tomography (CT) and to identify any factors that influence these results. MATERIALS AND METHODS: We retrospectively analyzed 439 consecutive patients with mild head and neck injuries. Mean HU values of the C2-C7 vertebra were determined on each sagittal, coronal, and axial CT image. Correlation patterns were analyzed between the HU value and corresponding dual-energy X-ray absorptiometry (DXA) in the lumbar vertebra (T-score) and femoral neck (T-score). A sub-group analysis was performed according to patient age, sex, and degree of spinal degeneration. RESULTS: The correlation coefficients for HU and DXA ranged from 0.52 to 0.65 in all cervical segments. A simple linear regression analysis revealed the following formula: T-score = 0.01 × (HU) - 4.55. The mean HU values for osteopenia and osteoporosis were 284.0 ± 63.3 and 231.5 ± 52.8, respectively. The ROC curve indicated that the HU method has a sensitivity of 89.2% and specificity of 88.7% to diagnose osteoporosis. The HU measurement showed a high correlation value (range: r = 0.64-0.70) with spine DXA score regardless of the degree of degeneration or patient age or sex. CONCLUSION: HU values using the upper two cervical vertebrae (C2 and C3) reflected a more reliable BMD level than other segments. Additionally, the HU of cervical CT provided reliable information regardless of measurement plane, age or sex, and degree of degeneration.

Analyzing the Significance of T1 Slope minus Cervical Lordosis in Patients with Anterior Cervical Discectomy and Fusion Surgery.

OBJECTIVE: Accurate measurement of T1 slope (a component of T1s minus cervical lordosis [CL]) is often constrained by anatomical limitations. In this situation, efforts should be made to find the exact meaning of T1s-CL and whether there are any alternatives to it. METHODS: We enrolled 117 patients who received two-level anterior cervical discectomy and fusion (ACDF). Occipital slope, C2 slope (C2s), C7 slope (C7s), T1, O-C2 angle (O-C2A), C2-7 angle (C2-7A), O-C7 angle (O-C7A), T1s-CL, C7-T1 angle (C7-T1A), and C2-7 sagittal vertical axis were measured. We determined 16° (T1s-CL) as the reference point for dividing subjects into the mismatch group and the balance group, and a comparative analysis was performed. RESULTS: The mean value of C7-T1A was constantly maintained within 2.6° peri-operatively. In addition, C2s and T1s-CL showed the same absolute change (Δ|0.8|°). The mean values of T1s-CL of the mismatch and balance groups were 23.0° and 7.6°, respectively. The five factors with the largest differences between the two groups were as follows : C2s (Δ13.3°), T1s-CL (Δ15.4°), O-C2A (Δ8.7°), C2-7A (Δ14.7°), and segmental angle (Δ7.9°) before surgery. Only four factors showed statistically significant change between the two groups after ACDF : T1s-CL (Δ4.0° vs. Δ0.2°), C2s (Δ3.2° vs. Δ0.7°), O-C2A (Δ2.6° vs. Δ1.3°), C2-7A (Δ6.3° vs. Δ1.3°). A very strong correlation between T1s-CL and C2s was also found (r=|0.88-0.96|). CONCLUSION: C2s itself may be the essential key to represent T1s-CL. The amounts and directions of change of these two factors (T1s-CL and C2s) were also almost identical. The above phenomenon was re-confirmed once again through the correlation analysis.

cAMP Response Element Binding-Protein- and Phosphorylation-Dependent Regulation of Tyrosine Hydroxylase by PAK4: Implications for Dopamine Replacement Therapy.

Parkinson's disease (PD) is characterized by a progressive loss of dopamine-producing neurons in the midbrain, which results in decreased dopamine levels accompanied by movement symptoms. Oral administration of l-3,4-dihydroxyphenylalanine (L-dopa), the precursor of dopamine, provides initial symptomatic relief, but abnormal involuntary movements develop later. A deeper understanding of the regulatory mechanisms underlying dopamine homeostasis is thus critically needed for the development of a successful treatment. Here, we show that p21-activated kinase 4 (PAK4) controls dopamine levels. Constitutively active PAK4 (caPAK4) stimulated transcription of tyrosine hydroxylase (TH) via the cAMP response element-binding protein (CREB) transcription factor. Moreover, caPAK4 increased the catalytic activity of TH through its phosphorylation of S40, which is essential for TH activation. Consistent with this result, in human midbrain tissues, we observed a strong correlation between phosphorylated PAK4S474, which represents PAK4 activity, and phosphorylated THS40, which reflects their enzymatic activity. Our findings suggest that targeting the PAK4 signaling pathways to restore dopamine levels may provide a new therapeutic approach in PD.

Significance of Cervical Spine Computed Tomography Hounsfield Units to Predict Bone Mineral Density and the Subsidence After Anterior Cervical Discectomy and Fusion.

STUDY DESIGN: This study was a retrospective review. OBJECTIVE: The purpose of this study was to investigate the correlation analysis between Hounsfield units (HU) and dual x-ray absorptiometry (DXA) based on the clinical results of patients who underwent anterior cervical discectomy and fusion (ACDF) surgery. SUMMARY OF BACKGROUND DATA: There is no technique to directly measure bone mineral density (BMD) in the cervical spine. As computed tomography is a very popular preoperative planning modality, using the HU value from that analysis to predict osteoporosis is important for patient outcomes and applications in the clinical field. MATERIALS AND METHODS: We reviewed the records for 235 patients who underwent 1-level (n=120) or 2-level (n=115) ACDF surgery. In the 1-level ACDF group, the HU was measured from C3 to C6 vertebra, while that for the 2-level ACDF group was measured from 3 surgical index vertebrae. The correlation patterns were analyzed with the corresponding DXA (T-score) for each patient. Subsidence of fusion segment was defined as change in distance between plate-tip and upper (lower) margin of the vertebra (index level) after 4 months of follow-up. In addition, to determine the relevant factors that influence fusion segment subsidence, other preoperative (C2 slope, C7 slope, C2-C7 angle, and C2-C7 sagittal vertical axis) and postoperative parameters (coronal angle and segmental angle change) were measured. RESULTS: The correlation coefficient between HU and DXA ranged from 0.57 to 0.71 in the 1-level ACDF group and from 0.59 to 0.66 in the 2-level ACDF group. The correlation between HU and DXA was statistically significant regardless of the degree of anterior osteophyte (r=-0.65 to 0.78). Total subsidence height was 3.8 mm after ACDF, and both HU and DXA were statistically correlated with total subsidence (r=0.26-0.28). In multivariate analysis, HU (middle vertebra) value was statistically associated with the degree of total subsidence. The high-subsidence group (≥4.5 mm) showed smaller HU values (284.1 vs. 316.0) and T-scores (-0.5 vs. 0.1) compared to the low-subsidence group (<4.5 mm). The discrepancy group, defined as cases with excess plate shift on 1 side, also showed smaller HU values (260.4 vs. 312.4) and higher degrees of total subsidence than the matched group. CONCLUSIONS: The correlation between HU in cervical computed tomography and lumbar DXA (T-score) was statistically significant in both 1-level and 2-level ACDF. The level of BMD (HU or DXA) is a very important factor for clinically determining the amount and regularity of subsidence after ACDF. Therefore, HU can be a good alternative assessment to accurately reflect as much of the BMD degree as DXA in the cervical spine.

Integrin-mediated adhesions in regulation of cellular senescence.

Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion-localized ßPAK-interacting exchange factor (ßPIX)-G protein-coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. ßPIX and GIT levels decline with age. ßPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of ßPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.

7 Tesla magnetic resonance imaging of caudal anterior cingulate and posterior cingulate cortex atrophy in patients with trigeminal neuralgia.

The cingulate cortex (CC) is a brain region that plays a key role in pain processing, but CC abnormalities are not unclear in patients with trigeminal neuralgia (TN). The purpose of this study was to determine the central causal mechanisms of TN and the surrounding brain structure in healthy controls and patients with TN using 7 Tesla (T) magnetic resonance imaging (MRI). Whole-brain parcellation in gray matter volume and thickness was assessed in 15 patients with TN and 16 healthy controls matched for sex, age, and regional variability using T1-weighted imaging. Regions of interest (ROIs) were measured in rostral anterior CC (rACC), caudal anterior CC (cACC) and posterior CC (PCC). We also investigated associations between gray matter volume or thickness and clinical symptoms, such as pain duration, Barrow Neurologic Institute (BNI) scores, offender vessel, and medications, in patients with TN. The cACC and PCC exhibited gray matter atrophy and reduced thickness between the TN and control groups. However, the rACC did not. Cortical volumes were negatively correlated with pain duration in transverse and inferior temporal areas, and thickness was also negatively correlated with pain duration in superior frontal and parietal areas. The cACC and PCC gray matter atrophy occurred in the patients with TN, and pain duration was associated with frontal, parietal, and temporal cortical regions. These results suggest that the cACC, PCC but not the rACC are associated with central pain mechanisms in TN.

7.0 Tesla MRI tractography in patients with trigeminal neuralgia.

7.0 Tesla (T) high-resolution diffusion tensor imaging (DTI) can supply information on changing microstructures in cranial nerves. We investigated DTI parameters and the feasibility of DTI criteria for diagnosing trigeminal neuralgia (TN). In this study, 14 patients (28 hemispheres) of mean age 49.0 years (range, 31-64) with TN underwent DTI using 7.0 TMRI. We compared fractional anisotropy (FA), axial diffusivity (AD), mean diffusivity (MD), and radial diffusivity (RD) of affected-side and unaffected-side trigeminal nerves using DTI. We examined associations between DTI parameters and clinical characteristics for patients with TN. In patients with TN, affected sides showed significantly decreased FA and significantly increased MD, and RD compared with unaffected sides of trigeminal nerves. Nuclei were not significantly different among patients with TN. Barrow Neurological Institute (BNI) pain scores did not correlate with affected sides. 7.0 T DTI was useful for detecting neurovascular compression in patients with TN. The increased signal-to-noise ratio provided by 7 T MRI should be advantageous for increasing spatial resolution to detect microstructure changes to trigeminal nerves in patients with TN.

Nigral dopaminergic PAK4 prevents neurodegeneration in rat models of Parkinson's disease.

Parkinson's disease (PD) is characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra. No neuroprotective treatments have successfully prevented the progression of this disease. We report that p21-activated kinase 4 (PAK4) is a key survival factor for DA neurons. We observed PAK4 immunoreactivity in rat and human DA neurons in brain tissue, but not in microglia or astrocytes. PAK4 activity was markedly decreased in postmortem brain tissue from PD patients and in rodent models of PD. Expression of constitutively active PAK4S445N/S474E (caPAK4) protected DA neurons in both the 6-hydroxydopamine and α-synuclein rat models of PD and preserved motor function. This neuroprotective effect of caPAK4 was mediated by phosphorylation of CRTC1 [CREB (adenosine 3',5'-monophosphate response element-binding protein)-regulated transcription coactivator] at S215. The nonphosphorylated form of CRTC1S215A compromised the ability of caPAK4 to induce the expression of the CREB target proteins Bcl-2, BDNF, and PGC-1α. Our results support a neuroprotective role for the PAK4-CRTC1S215-CREB signaling pathway and suggest that this pathway may be a useful therapeutic target in PD.

p21-activated kinase 4 critically regulates melanogenesis via activation of the CREB/MITF and ß-catenin/MITF pathways.

p21-activated kinase 4 (PAK4) regulates a wide range of cellular events, including cytoskeletal remodeling, cell growth, and survival. Our previous study identified PAK4 as a key regulator of cAMP-response element-binding protein (CREB) that acts upstream of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. We therefore investigated the role of PAK4 in melanogenesis. Melanocytes express both PAK2 and PAK4 isoforms, but only RNA interference knockdown of PAK4 significantly influenced α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in B16 melanoma cells. Consistent with this result, PAK4 inhibition by PF3758309, a potent ATP-competitive inhibitor of PAKs, suppressed not only α-MSH-induced melanogenesis in B16 melanoma and human epithelial melanocyte cells but also UVB-induced melanogenesis in the skin of melanin-possessing hairless mice (HRM-2) in a dose-dependent manner. Inhibition of PAK4 over several days markedly decreased the levels of CREB, MITF, and tyrosinase in both HRM-2 mice and B16 melanoma cells. Moreover, PAK4 knockdown and inhibition suppressed α-MSH-stimulated ß-catenin phosphorylation at serine 675 (S675) but enhanced phosphorylation at S33/37, an indicator for ubiquitination-dependent proteolysis. Together, our results provide evidence that PAK4 promotes α-MSH/UVB-induced melanogenesis via the CREB and Wnt/ß-catenin signaling pathways and suggest that PAK4 may be a potential therapeutic target in pigmentation disorders.

Phosphorylation of ß-catenin at serine 663 regulates its transcriptional activity.

ß-Catenin, a component of Wnt signaling, plays a key role in colorectal carcinogenesis. The phosphorylation status of ß-catenin determines its fate and affects its cellular function, and serine 675 (S675) was previously identified as a common target of p21-activated kinase 1 (PAK1) and protein kinase A. In the present study, we explored the PAK1-specific phosphorylation site(s) in ß-catenin. Active PAK1 T423E but not inactive PAK1 K299R interacted with and phosphorylated ß-catenin. Mutagenesis followed by a kinase assay revealed that PAK1 phosphorylated S663 in addition to S675, and an anti-phospho-ß-catenin(S663) antibody detected the phosphorylation of S663 downstream of PAK1 in various human colon cancer cells. Furthermore, the Wnt3a-stimulated S663 phosphorylation was inhibited by the PAK1-specific inhibitor, IPA-3, but not by H-89 or LY294002. The non-phosphorylatable mutant forms of ß-catenin, S663A, S675A and S663/675A, showed similar defects in their PAK1-induced TCF/LEF transactivation, whereas the phosphomimetic form of ß-catenin, S663D, demonstrated a transcriptional activity that was comparable to that of ß-catenin S675D and ß-catenin S663D/S675D. Taken together, these results provide evidence that PAK1 specifically phosphorylates ß-catenin at S663 and that this phosphorylation is essential for the PAK1-mediated transcriptional activation of ß-catenin.