Molecular characterization, expression, and functional analysis of cystatin B in the big-belly seahorse (Hippocampus abdominalis).

Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses.

Cystatin B-deficiency triggers ectopic histone H3 tail cleavage during neurogenesis.

Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

Cross Talk at the Cytoskeleton-Plasma Membrane Interface: Impact on Neuronal Morphology and Functions.

The cytoskeleton and its associated proteins present at the plasma membrane not only determine the cell shape but also modulate important aspects of cell physiology such as intracellular transport including secretory and endocytic pathways. Continuous remodeling of the cell structure and intense communication with extracellular environment heavily depend on interactions between cytoskeletal elements and plasma membrane. This review focuses on the plasma membrane-cytoskeleton interface in neurons, with a special emphasis on the axon and nerve endings. We discuss the interaction between the cytoskeleton and membrane mainly in two emerging topics of neurobiology: (i) production and release of extracellular vesicles and (ii) local synthesis of new proteins at the synapses upon signaling cues. Both of these events contribute to synaptic plasticity. Our review provides new insights into the physiological and pathological significance of the cytoskeleton-membrane interface in the nervous system.

Plasma Cystatin B Association With Abdominal Aortic Aneurysms and Need for Later Surgical Repair: A Sub-study of the VIVA Trial.

OBJECTIVE/BACKGROUND: The development of an abdominal aortic aneurysm (AAA) involves extensive extracellular matrix remodelling, leading to aortic wall weakening. This process is mediated by proteases, including cysteinyl cathepsins. Cystatins are their endogenous inhibitors. This study tested whether plasma cystatin B levels in patients with AAA differed from those of healthy controls. METHODS: Plasma samples from patients with AAA and age matched controls were selected from the Viborg Vascular (VIVA) screening trial for AAA. Enzyme linked immunosorbent assay determined plasma cystatin B. T-test, logistic regression, Pearson's correlation and Cox regression tested whether plasma cystatin B correlates with AAA size and growth rate, or serves as a marker for AAA. RESULTS: Plasma cystatin B levels were significantly higher in patients with AAA than in controls (p < 0.001). Logistic regression analysis showed that cystatin B tertile at baseline was associated with the presence of AAA before (odds ratio [OR] 1.656; p < 0.001) and after adjustment for peripheral arterial disease (PAD), chronic obstructive pulmonary disease (COPD), and previous ischaemic events (OR 1.526; p < 0.001). A t-test showed a significant association between cystatin B and PAD at screening, hospital diagnosis of COPD, previous atherosclerotic events, and use of low dose aspirin. Pearson's correlation test showed positive and significant associations between cystatin B and AAA size (r = 0.15; p < 0.001). Cox regression test showed that plasma cystatin B tertile at baseline was associated with later AAA surgical repair before (hazard ratio [HR] 1.387; p < 0.001) and after adjustment for PAD, COPD, previous ischaemic event, and maximum infrarenal aortic diameter (HR 1.523; p < 0.001). CONCLUSION: In contrast to prior studies that showed that cystatin C is negatively associated with AAA development, this study demonstrated a positive association between cystatin B and AAA size and associations between cystatin B tertile at baseline and AAA presence and need for later surgical repair. It is possible that these two cystatins inhibit cathepsin activity and participate in AAA with different mechanisms.

Molecular characterization, expression and immune functions of cystatin B in Japanese flounder (Paralichthys olivaceus).

Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder (Paralichthys olivaceus) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1ß and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts.

Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.

Brain inflammation is accompanied by peripheral inflammation in Cstb -/- mice, a model for progressive myoclonus epilepsy.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb -/- ) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb -/- mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb -/- mice and higher CXCL13 expression in activated microglia in Cstb -/- compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb -/- mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb -/- mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.

Cystatin B and HIV regulate the STAT-1 signaling circuit in HIV-infected and INF-ß-treated human macrophages.

Cystatin B is a cysteine protease inhibitor that induces HIV replication in monocyte-derived macrophages (MDM). This protein interacts with signal transducer and activator of transcription (STAT-1) factor and inhibits the interferon (IFN-ß) response in Vero cells by preventing STAT-1 translocation to the nucleus. Cystatin B also decreases the levels of tyrosine-phosphorylated STAT-1 (STAT-1PY). However, the mechanisms of cystatin B regulation on STAT-1 phosphorylation in MDM are unknown. We hypothesized that cystatin B inhibits IFN-ß antiviral responses and induces HIV replication in macrophage reservoirs through the inhibition of STAT-1 phosphorylation. Macrophages were transfected with cystatin B siRNA prior to interferon-ß treatment or infected with HIV-ADA to determine the effect of cystatin B modulation in STAT-1 localization and activation using immunofluorescence and proximity ligation assays. Cystatin B decreased STAT-1PY and its transportation to the nucleus, while HIV infection retained unphosphorylated STAT (USTAT-1) in the nucleus avoiding its exit to the cytoplasm for eventual phosphorylation. In IFN-ß-treated MDM, cystatin B inhibited the nuclear translocation of both, USTAT-1 and STAT-1PY. These results demonstrate that cystatin B interferes with the STAT-1 signaling and IFN-ß-antiviral responses perpetuating HIV in macrophage reservoirs.

Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and oxidative stress.

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.

Abnormal microglial activation in the Cstb(-/-) mouse, a model for progressive myoclonus epilepsy, EPM1.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal-recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss-of-function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb(-/-) mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb(-/-) mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb(-/-) mouse brain, expression of the inflammatory marker p-p38 MAPK and the proportion of both pro-inflammatory M1 and anti-inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb(-/-) mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro-inflammatory inducible nitric oxide synthase (iNOS) and anti-inflammatory arginase 1 (ARG1) in Cstb(-/-) mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb(-/-) microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1.

No evidence of a role for cystatin B gene in juvenile myoclonic epilepsy.

Genetic factors play a major role in the etiology of juvenile myoclonic epilepsy (JME), a common form of idiopathic generalized epilepsy, but so far, genes related to JME remain largely unknown. JME shares electroclinical features with Unverricht-Lundborg disease (progressive myoclonic epilepsy type 1; EPM1), a form of progressive myoclonus epilepsy characterized by myoclonus, epilepsy, and gradual neurologic deterioration. EPM1 is caused by mutations in the gene that codes for cystatin B (CSTB), an inhibitor of cysteine protease. In the present study, we wished to investigate the role of the CSTB gene in patients with JME. Fifty-seven unrelated patients (35 women; mean age ± standard deviation [SD], 24.1 ± 7.7; mean age ± SD at onset, 15.3 ± 2.4) with JME were enrolled. Twenty-three of 57 patients were the probands of families with JME. The molecular diagnosis was carried out to identify the common dodecamer repeat expansion mutation or other disease-causing mutations in the CSTB gene. The molecular analysis did not depict mutations in any of the 57 patients with JME. Our study did not support a role for the CSTB gene in patients with familial or sporadic JME.

Insights in progressive myoclonus epilepsy: HSP70 promotes cystatin B polymerization.

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu(2+) for chaperone-dependent polymerization. Cu(2+) interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu(2+) modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu(2+)/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s).

The Roles of Cystatin B in the Brain and Pathophysiological Mechanisms of Progressive Myoclonic Epilepsy Type 1.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene (CSTB). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Biomarker cystatin B expression correlates with pathogenesis in cervical cancer.

OBJECTIVE: Cervical cancer (CC) is one of the most common gynecologic malignancies worldwide. Although rapid improvements have been made regarding its prevention and treatment, little is known about disease pathogenesis and the clinical relevance of reliable biomarkers. The present study evaluated the expression of cystatin B (CSTB) as a potential biomarker of CC. METHODS: Tissue microarray analysis and immunohistochemical staining were performed to detect CSTB expression, while CSTB mRNA and protein expression levels of freshly isolated CC tissue were measured by quantitative real-time PCR and western blot, respectively. Bioinformatics were used to analyze the CSTB co-expression network and functional enrichments. RESULTS: We observed high CSTB mRNA and protein expression levels in CC tissues, which was confirmed by tissue microarray in a comparison with paired adjacent non-cancerous cervical tissue samples. CSTB gene enrichments and associations with co-expressed genes were also observed. Further analysis showed that elevated CSTB expression was associated with pathological progress in CC. CONCLUSION: Our data demonstrate that CSTB has the potential to be used as a tissue biomarker with clinical value in patients with CC, which may aid the development of intervention strategies.

A Clinical Study on Urinary Clusterin and Cystatin B in Dogs with Spontaneous Acute Kidney Injury.

Novel biomarkers are needed in diagnosing reliably acute kidney injury (AKI) in dogs and in predicting morbidity and mortality after AKI. Our hypothesis was that two novel tubular biomarkers, urinary clusterin (uClust) and cystatin B (uCysB), are elevated in dogs with AKI of different etiologies. In a prospective, longitudinal observational study, we collected serum and urine samples from 18 dogs with AKI of different severity and of various etiology and from 10 healthy control dogs. Urinary clusterin and uCysB were compared at inclusion between dogs with AKI and healthy controls and remeasured one and three months later. Dogs with AKI had higher initial levels of uClust (median 3593 ng/mL; interquartile range [IQR]; 1489-10,483) and uCysB (554 ng/mL; 29-821) compared to healthy dogs (70 ng/mL; 70-70 and 15 ng/mL; 15-15; p < 0.001, respectively). Initial uCysB were higher in dogs that died during the one-month follow-up period (n = 10) (731 ng/mL; 517-940), compared to survivors (n = 8) (25 ng/mL; 15-417 (p = 0.009). Based on these results, uClust and especially uCysB are promising biomarkers of AKI. Further, they might reflect the severity of tubular injury, which is known to be central to the pathology of AKI.

Cathepsin B abundance, activity and microglial localisation in Alzheimer's disease-Down syndrome and early onset Alzheimer's disease; the role of elevated cystatin B.

Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimer's disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimer's disease (EOAD). The impact of the additional copy of CSTB on Alzheimer's disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and CSTB in individuals who had Down syndrome and Alzheimer's disease, with disomic individuals who had Alzheimer's disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimer's disease compared with disomic individuals who had Alzheimer's disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B+ cells. We find that the abundance of CSTB is significantly increased in the brains of individuals who have Down syndrome and Alzheimer's disease compared to disomic individuals both with and without Alzheimer's disease. In mouse and human cellular preclinical models of Down syndrome, three-copies of CSTB increases CSTB protein abundance but this is not sufficient to modulate cathepsin B activity. EOAD and Alzheimer's disease-Down syndrome share many overlapping mechanisms but differences in disease occur in individuals who have trisomy 21. Understanding this biology will ensure that people who have Down syndrome access the most appropriate Alzheimer's disease therapeutics and moreover will provide unique insight into disease pathogenesis more broadly.

Pathological Deficit of Cystatin B Impairs Synaptic Plasticity in EPM1 Human Cerebral Organoids.

Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology.

Characterization of Cystatin B Interactome in Saliva from Healthy Elderly and Alzheimer's Disease Patients.

Cystatin B is a small, multifunctional protein involved in the regulation of inflammation, innate immune response, and neuronal protection and found highly abundant in the brains of patients with Alzheimer's disease (AD). Recently, our study demonstrated a significant association between the level of salivary cystatin B and AD. Since the protein is able to establish protein-protein interaction (PPI) in different contexts and aggregation-prone proteins and the PPI networks are relevant for AD pathogenesis, and due to the relevance of finding new AD markers in peripheral biofluids, we thought it was interesting to study the possible involvement of cystatin B in PPIs in saliva and to evaluate differences and similarities between AD and age-matched elderly healthy controls (HC). For this purpose, we applied a co-immunoprecipitation procedure and a bottom-up proteomics analysis to purify, identify, and quantify cystatin B interactors. Results demonstrated for the first time the existence of a salivary cystatin B-linked multi-protein complex composed by 82 interactors and largely expressed in the body. Interactors are involved in neutrophil activation, antimicrobial activity, modulation of the cytoskeleton and extra-cellular matrix (ECM), and glucose metabolism. Preliminary quantitative data showed significantly lower levels of triosophosphate isomerase 1 and higher levels of mucin 7, BPI, and matrix Gla protein in AD with respect to HC, suggesting implications associated with AD of altered glucose metabolism, antibacterial activities, and calcification-associated processes. Data are available via ProteomeXchange with identifiers PXD039286 and PXD030679.

In depth behavioral phenotyping unravels complex motor disturbances in Cstb-/- mouse, a model for progressive myoclonus epilepsy type 1.

Progressive myoclonus epilepsy type 1 (EPM1) is an autosomal recessively inherited childhood-adolescence onset neurodegenerative disease caused by mutations in the cystatin B (CSTB gene). The key clinical manifestation in EPM1 is progressive, stimulus-sensitive, in particular action-induced myoclonus. The cystatin B-deficient mouse model, Cstb-/-, has been described to present with myoclonic seizures and progressive ataxia. Here we describe results from in-depth behavioral phenotyping of the Cstb-/- mouse model in pure isogenic 129S2/SvHsd background covering ages from 1.5 to 6 months. We developed a method for software-assisted detection of myoclonus from video recordings of the Cstb-/- mice. Additionally, we observed that the mice were hyperactive and showed reduced startle response, problems in motor coordination and lack of inhibition. We were, however, not able to demonstrate an ataxic phenotype in them. This detailed behavioral phenotyping of the Cstb-/- mice reveals new aspects of this mouse model. The nature of the motor problems in the Cstb-/- mice seems to be more complex and more resembling the human phenotype than initially described.