The Crocus sativus Compounds trans-Crocin 4 and trans-Crocetin Modulate the Amyloidogenic Pathway and Tau Misprocessing in Alzheimer Disease Neuronal Cell Culture Models

Crocus sativus L. natural compounds have been extensively used in traditional medicine for thousands of years. Recent research evidence is now emerging in support of its therapeutic potential for different pathologies including neurodegenerative diseases. Herein, the C. sativus L. natural compounds trans-crocin 4 and trans-crocetin were selected for in depth molecular characterization of their potentially protective effects against Alzheimer’s Disease (AD), utilizing two AD neuronal cell culture models (SH-SY5Y overexpressing APP and PC12 expressing hyperphosphorylated tau). Biologically relevant concentrations, ranging from 0.1 μM to 1 mM, applied for 24 h or 72 h, were well tolerated by differentiated wild type SH-SY5Y and PC12 cells. When tested on neuronally differentiated SH-SY5Y-APP both trans-crocin 4 and trans-crocetin had significant effects against amyloidogenic pathways. Trans-crocin 4 significantly decreased of β-secretase, a key enzyme of the amyloidogenic pathway, and APP-C99, while it decreased γ-secretases that generate toxic beta-amyloid peptides. Similarly, trans-crocetin treatment led to a reduction in β- and γ-secretases, as well as to accumulation of cellular AβPP. When tested on the neuronally differentiated PC12-htau cells, both compounds proved effective in suppressing the active forms of GSK3β and ERK1/2 kinases, as well as significantly reducing total tau and tau phosphorylation. Collectively, our data demonstrate a potent effect of trans-crocin 4 and trans-crocetin in suppressing key molecular pathways of AD pathogenesis, rendering them a promising tool in the prevention and potentially the treatment of AD

Identification of a protein phosphatase-1/phospholamban complex that is regulated by cAMP-dependent phosphorylation.

In human and experimental heart failure, the activity of the type 1 phosphatase is significantly increased, associated with dephosphorylation of phospholamban, inhibition of the sarco(endo)plasmic reticulum Ca(2+) transport ATPase (SERCA2a) and depressed function. In the current study, we investigated the molecular mechanisms controlling protein phosphatase-1 activity. Using recombinant proteins and complementary in vitro binding studies, we identified a multi-protein complex centered on protein phosphatase-1 that includes its muscle specific glycogen-targeting subunit GM and substrate phospholamban. GM interacts directly with phospholamban and this association is mediated by the cytosolic regions of the proteins. Our findings suggest the involvement of GM in mediating formation of the phosphatase-1/GM/phospholamban complex through the direct and independent interactions of GM with both protein phosphatase-1 and phospholamban. Importantly, the protein phosphatase-1/GM/phospholamban complex dissociates upon protein kinase A phosphorylation, indicating its significance in the β-adrenergic signalling axis. Moreover, protein phosphatase-1 activity is regulated by two binding partners, inhibitor-1 and the small heat shock protein 20, Hsp20. Indeed, human genetic variants of inhibitor-1 (G147D) or Hsp20 (P20L) result in reduced binding and inhibition of protein phosphatase-1, suggesting aberrant enzymatic regulation in human carriers. These findings provide insights into the mechanisms underlying fine-tuned regulation of protein phosphatase-1 and its impact on the SERCA2/phospholamban interactome in cardiac function

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca²⁺ Handling and Arrhythmogenesis

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients

The Cardioprotective PKA-Mediated Hsp20 Phosphorylation Modulates Protein Associations Regulating Cytoskeletal Dynamics

The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology

Hsp20 variant (P20L) variant exhibits diminished binding and inhibition of PP1.

<p>(A) SDS-gel stained with Coomassie blue showing purified GST-Hsp20 WT and P20L recombinant proteins. (B) Blot overlay assay with MBP-PP1 protein and immunodetection with MBP antibody demonstrates reduced PP1 binding to P20L protein. Phosphorylation of recombinant Hsp20 proteins by PKA treatment resulted in increased binding of PP1 to Hsp20-WT, however, this effect was not observed for P20L variant. (C) Quantification of PP1 protein binding to WT and P20L under basal and PKA conditions. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). (D) Hsp20 variant P20L presents diminished phosphorylation levels at Ser16. (E) PP1 activity measurements showed reduced inhibition of PP1 by P20L. n = 4; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs WT (–PKA); #<i>P</i><0.05 vs WT (+PKA). WB: Western blot.</p

Schematic Representation of PP1 protein complexes regulating PLN phosphorylation and cardiac contractility.

<p>A regulatory PP1 signaling complex controls PLN phosphorylation. (A) Under basal conditions, G_M associates with both PP1 and PLN, enabling effective targeting of the phosphatase to its substrate. (B) Following β-adrenergic stimulation, protein phosphorylation leads to dissociation of the PP1/G_M/PLN complex, enabling PP1 to bind with high affinity to its regulatory proteins Inhibitor-1 and Hsp20. This results in inhibition of PP1 and enhanced phosphorylation of PLN as well as increased cardiac contractility.</p

G_M phosphorylation inhibits its interactions with PLN and PP1.

<p>(A) Blot overlay assays on MBP-G_M (amino acid 382-778) recombinant protein was performed using non-phosphorylated or PKA-phosphorylated GST-PLN protein. Western blot analysis revealed decreased binding of phosphorylated PLN to G_M. (B) Quantification of G_M and PLN binding established a significant reduction upon PLN phosphorylation. n = 6; <i>t</i>-test, two-tailed, *<i>P</i><0.05 vs –PKA. (C) Pull down assays using GST-G_M (amino acid 382-778) recombinant protein and phosphorylated or non-phosphorylated cardiac homogenates illustrated the reduced association of PLN with G_M in the PKA-phosphorylated sample. (D) Blot overlay using phosphorylated or non-phosphorylated MBP-G_M (amino acid 1-386) revealed the lack of PP1 binding to phosphorylated G_M protein.</p