Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies.

Neurodevelopmental proteasomopathies represent a distinctive category of neurodevelopmental disorders (NDD) characterized by genetic variations within the 26S proteasome, a protein complex governing eukaryotic cellular protein homeostasis. In our comprehensive study, we identified 23 unique variants in PSMC5 , which encodes the AAA-ATPase proteasome subunit PSMC5/Rpt6, causing syndromic NDD in 38 unrelated individuals. Overexpression of PSMC5 variants altered human hippocampal neuron morphology, while PSMC5 knockdown led to impaired reversal learning in flies and loss of excitatory synapses in rat hippocampal neurons. PSMC5 loss-of-function resulted in abnormal protein aggregation, profoundly impacting innate immune signaling, mitophagy rates, and lipid metabolism in affected individuals. Importantly, targeting key components of the integrated stress response, such as PKR and GCN2 kinases, ameliorated immune dysregulations in cells from affected individuals. These findings significantly advance our understanding of the molecular mechanisms underlying neurodevelopmental proteasomopathies, provide links to research in neurodegenerative diseases, and open up potential therapeutic avenues.

IRAK3-mediated suppression of pro-inflammatory MyD88/IRAK signaling affects disease severity in acute pancreatitis.

Acute pancreatitis (AP), which is characterized by self-digestion of the pancreas by its own prematurely activated digestive proteases, is a major reason for hospitalization. The autodigestive process causes necrotic cell death of pancreatic acinar cells and the release of damage associated molecular pattern which activate macrophages and drive the secretion of pro-inflammatory cytokines. The MYD88/IRAK signaling pathway plays an important role for the induction of inflammatory responses. Interleukin-1 receptor associated kinase-3 (IRAK3) is a counter-regulator of this pathway. In this study, we investigated the role of MYD88/IRAK using Irak3-/- mice in two experimental animal models of mild and severe AP. IRAK3 is expressed in macrophages as well as pancreatic acinar cells where it restrains NFκB activation. Deletion of IRAK3 enhanced the migration of CCR2+ monocytes into the pancreas and triggered a pro-inflammatory type 1 immune response characterized by significantly increased serum levels of TNFα, IL-6, and IL-12p70. Unexpectedly, in a mild AP model this enhanced pro-inflammatory response resulted in decreased pancreatic damage, whereas in a severe AP model, induced by partial pancreatic duct ligation, the increased pro-inflammatory response drives a severe systemic inflammatory response syndrome (SIRS) and is associated with an increased local and systemic damage. Our results indicate that complex immune regulation mechanism control the course of AP, where moderate pro-inflammation not necessarily associates with increased disease severity but also drives tissue regenerative processes through a more effective clearance of necrotic acinar cells. Only when the pro-inflammation exceeds a certain systemic level, it fuels SIRS and increases disease severity.

Novel p.G250A Mutation Associated with Chronic Pancreatitis Highlights Misfolding-Prone Region in Carboxypeptidase A1 (CPA1).

Inborn mutations in the digestive protease carboxypeptidase A1 (CPA1) gene may be associated with hereditary and idiopathic chronic pancreatitis (CP). Pathogenic mutations, such as p.N256K, cause intracellular retention and reduced secretion of CPA1, accompanied by endoplasmic reticulum (ER) stress, suggesting that mutation-induced misfolding underlies the phenotype. Here, we report the novel p.G250A CPA1 mutation found in a young patient with CP. Functional properties of the p.G250A mutation were identical to those of the p.N256K mutation, confirming its pathogenic nature. We noted that both mutations are in a catalytically important loop of CPA1 that is stabilized by the Cys248-Cys271 disulfide bond. Mutation of either or both Cys residues to Ala resulted in misfolding, as judged by the loss of CPA1 secretion and intracellular retention. We re-analyzed seven previously reported CPA1 mutations that affect this loop and found that all exhibited reduced secretion and caused ER stress of varying degrees. The magnitude of ER stress was proportional to the secretion defect. Replacing the naturally occurring mutations with Ala (e.g., p.V251A for p.V251M) restored secretion, with the notable exception of p.N256A. We conclude that the disulfide-stabilized loop of CPA1 is prone to mutation-induced misfolding, in most cases due to the disruptive nature of the newly introduced side chain. We propose that disease-causing CPA1 mutations exhibit abolished or markedly reduced secretion with pronounced ER stress, whereas CPA1 mutations with milder misfolding phenotypes may be associated with lower disease risk or may not be pathogenic at all.

A rare PRSS1 p.S127C mutation is associated with chronic pancreatitis and causes misfolding-induced ER-stress.

BACKGROUND: /Objectives: Sequence variants in several genes have been identified as being associated with an increased inherited risk to develop chronic pancreatitis (CP). In a genetic survey of a CP patient we identified in the PRSS1gene a new c.380C > G sequence variation, giving rise to a non-synonymous p.S127C mutation. Functional studies were performed to analyze the associated pathophysiology of the variant. METHODS: Following generation of an expression vector for the new PRSS1 variant we compared its expression, secretion and catalytic activity with already known PRSS1 risk variants in HEK 293T cells. The intracellular protein accumulation and induction of endoplasmic reticulum (ER)-stress was analyzed. RESULTS: Prediction tool analysis indicated a probably deleterious effect of the p.S127C variant on protein function which was confirmed by detection of a secretion defect in HEK293T cells leading to intracellular protein accumulation. While protein misfolding was associated with reduced trypsin activity, the increased expression of BIP and presence of spliced XBP1 indicated that the p.S127C variant induces ER stress and activates the UPR signaling pathway. CONCLUSIONS: The disease mechanism of the PRSS1 p.S127C variant involves defective protein secretion and the induction of ER-stress due to accumulation of presumably misfolded trypsinogen within the ER. The new variant should be considered disease-causing with an incomplete penetrance. Our results confirm that in addition to dysregulated trypsin-activity or reduced fluid secretion, ER-stress induction is an important trigger for acinar cell damage and the development of recurrent or chronic pancreatic inflammation.

Role of Bile Acids and Bile Salts in Acute Pancreatitis: From the Experimental to Clinical Studies.

ABSTRACT: Acute pancreatitis (AP) is one of the most common gastroenterological disorders leading to hospitalization. It has long been debated whether biliary AP, about 30% to 50% of all cases, is induced by bile acids (BAs) when they reach the pancreas via reflux or via the systemic blood circulation.Besides their classical function in digestion, BAs have become an attractive research target because of their recently discovered property as signaling molecules. The underlying mechanisms of BAs have been investigated in various studies. Bile acids are internalized into acinar cells through specific G-protein-coupled BA receptor 1 and various transporters. They can further act via different receptors: the farnesoid X, ryanodine, and inositol triphosphate receptor. Bile acids induce a sustained Ca2+ influx from the endoplasmic reticulum and release of Ca2+ from acidic stores into the cytosol of acinar cells. The overload of intracellular Ca2+ results in mitochondrial depolarization and subsequent acinar cell necrosis. In addition, BAs have a biphasic effect on pancreatic ductal cells. A more detailed characterization of the mechanisms through which BAs contribute to the disease pathogenesis and severity will greatly improve our understanding of the underlying pathophysiology and may allow for the development of therapeutic and preventive strategies for gallstone-inducedAP.

Early trypsin activation develops independently of autophagy in caerulein-induced pancreatitis in mice.

Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile-Pro-Arg-AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.

Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas.

The endoplasmic reticulum (ER) is the site of synthesis and folding of membrane and secretory proteins. The fraction of protein passing through the ER represents a large proportion of the total protein in the cell. Protein folding, glycosylation, sorting and transport are essential tasks of the ER and a compromised ER folding network has been recognized to be a key component in the disease pathogenicity of common neurodegenerative, metabolic and malignant diseases. On the other hand, the ER protein folding machinery also holds significant potential for therapeutic interventions. Many causes can lead to ER stress. A disturbed calcium homeostasis, the generation of reactive oxygen species (ROS) and a persistent overload of misfolded proteins within the ER can drive the course of adisease. In this review the role of ER-stress in diseases of the liver and pancreas will be examined using pancreatitis and Wilson´s disease as examples. Potential therapeutic targets in ER-stress pathways will also be discussed.

[Feasibility and effects of a psychotherapeutic group intervention for caregiving relatives of people with dementia]. / Machbarkeit und Effekte einer psychotherapeutischen Gruppenintervention für pflegende Angehörige von Menschen mit Demenz.

INTRODUCTION: Family caregivers of people with dementia (PwD) have a high burden and therefore are themselves at a high risk for psychiatric and somatic morbidities. Although individual psychotherapy has been shown to be a potentially effective treatment, it is rarely used by family caregivers. Possible reasons are poor accessibility and time restrictions on the side of the caregiver. AIM: To test the efficacy of a short-term and low threshold psychotherapeutic group intervention for family caregivers of PwD with respect to mental stability of the caregivers. MATERIAL AND METHODS: Data from a 12-week psychotherapeutic group intervention (10 participants each in the intervention and control groups) were analyzed. Main topics of the intervention were: personal limits, dysfunctional thoughts, emotions and resource activation. Primary endpoints were an increase of perceived self-efficacy and reduction of depressive symptoms using SWE and ADS questionnaires before, directly and 3 months after the end of the intervention. RESULTS: A gain in perceived self-efficacy did not reach statistical significance, whereas depressive symptoms showed a statistically significant increase in the intervention group over time compared to the control group. DISCUSSION: The intervention did not reach its primary endpoints. Possible reasons are the fact that the group was highly heterogeneous with respect to dementia etiology and the low number of participants. The short duration of the intervention may have reduced the potential of the program to address all urgent needs of the participants.