Functional Signature of LRP4 Antibodies in Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Antibodies (Abs) specific for the low-density lipoprotein receptor-related protein 4 (LRP4) occur in up to 5% of patients with myasthenia gravis (MG). The objective of this study was to profile LRP4-Ab effector actions. METHODS: We evaluated the efficacy of LRP4-specific compared with AChR-specific IgG to induce Ab-dependent cellular phagocytosis (ADCP), Ab-dependent cellular cytotoxicity (ADCC), and Ab-dependent complement deposition (ADCD). Functional features were additionally assessed in an independent AChR-Ab+ MG cohort. Levels of circulating activated complement proteins and frequency of Fc glycovariants were quantified and compared with demographically matched 19 healthy controls. RESULTS: Effector actions that required binding of Fc domains to cellular FcRs such as ADCC and ADCP were detectable for both LRP4-specific and AChR-specific Abs. In contrast to AChR-Abs, LRP4-binding Abs showed poor efficacy in inducing complement deposition. Levels of circulating activated complement proteins were not substantially increased in LRP4-Ab-positive MG. Frequency of IgG glycovariants carrying 2 sialic acid residues, indicative for anti-inflammatory IgG activity, was decreased in patients with LRP4-Ab-positive MG. DISCUSSION: LRP4-Abs are more effective in inducing cellular FcR-mediated effector mechanisms than Ab-dependent complement activation. Their functional signature is different from AChR-specific Abs.

LRP10 and α-synuclein transmission in Lewy body diseases.

Autosomal dominant variants in LRP10 have been identified in patients with Lewy body diseases (LBDs), including Parkinson's disease (PD), Parkinson's disease-dementia (PDD), and dementia with Lewy bodies (DLB). Nevertheless, there is little mechanistic insight into the role of LRP10 in disease pathogenesis. In the brains of control individuals, LRP10 is typically expressed in non-neuronal cells like astrocytes and neurovasculature, but in idiopathic and genetic cases of PD, PDD, and DLB, it is also present in α-synuclein-positive neuronal Lewy bodies. These observations raise the questions of what leads to the accumulation of LRP10 in Lewy bodies and whether a possible interaction between LRP10 and α-synuclein plays a role in disease pathogenesis. Here, we demonstrate that wild-type LRP10 is secreted via extracellular vesicles (EVs) and can be internalised via clathrin-dependent endocytosis. Additionally, we show that LRP10 secretion is highly sensitive to autophagy inhibition, which induces the formation of atypical LRP10 vesicular structures in neurons in human-induced pluripotent stem cells (iPSC)-derived brain organoids. Furthermore, we show that LRP10 overexpression leads to a strong induction of monomeric α-synuclein secretion, together with time-dependent, stress-sensitive changes in intracellular α-synuclein levels. Interestingly, patient-derived astrocytes carrying the c.1424 + 5G > A LRP10 variant secrete aberrant high-molecular-weight species of LRP10 in EV-free media fractions. Finally, we show that this truncated patient-derived LRP10 protein species (LRP10splice) binds to wild-type LRP10, reduces LRP10 wild-type levels, and antagonises the effect of LRP10 on α-synuclein levels and distribution. Together, this work provides initial evidence for a possible functional role of LRP10 in LBDs by modulating intra- and extracellular α-synuclein levels, and pathogenic mechanisms linked to the disease-associated c.1424 + 5G > A LRP10 variant, pointing towards potentially important disease mechanisms in LBDs.

Functional characterization of SORL1 variants in cell-based assays to investigate variant pathogenicity.

SORLA, the protein encoded by the SORL1 gene, has an important role in recycling cargo proteins to the cell surface. While SORLA loss-of-function variants occur almost exclusively in Alzheimer's disease cases, the majority of SORL1 variants are missense variants that are individually rare and can have individual mechanisms how they impair SORLA function as well as have individual effect size on disease risk. However, since carriers mostly come from small pedigrees, it is challenging to determine variant penetrance, leaving clinical significance associated with most missense variants unclear. In this article, we present functional approaches to evaluate the pathogenicity of a SORL1 variant, p.D1105H. First, we generated our mutant receptor by inserting the D1105H variant into the full-length SORLA-WT receptor. Then using western blot analysis we quantified the effect of the mutation on maturation and shedding of the receptor for transfected cells, and finally applied a flow cytometry approach to quantify SORLA expression at the cell surface. The results showed decreased maturation, decreased shedding, and decreased cell surface expression of D1105H compared with wild-type SORLA. We propose how these approaches can be used to functionally assess the pathogenicity of SORL1 variants in the future. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Differential effects of SORL1 deficiency on the endo-lysosomal network in human neurons and microglia.

The endosomal gene SORL1 is a strong Alzheimer's disease (AD) risk gene that harbours loss-of-function variants causative for developing AD. The SORL1 protein SORL1/SORLA is an endosomal receptor that interacts with the multi-protein sorting complex retromer to traffic various cargo through the endo-lysosomal network (ELN). Impairments in endo-lysosomal trafficking are an early cellular symptom in AD and a novel therapeutic target. However, the cell types of the central nervous system are diverse and use the ELN differently. If this pathway is to be effectively therapeutically targeted, understanding how key molecules in the ELN function in various cell types and how manipulating them affects cell-type specific responses relative to AD is essential. Here, we discuss an example where deficiency of SORL1 expression in a human model leads to stress on early endosomes and recycling endosomes in neurons, but preferentially leads to stress on lysosomes in microglia. The differences observed in these organelles could relate to the unique roles of these cells in the brain as neurons are professional secretory cells and microglia are professional phagocytic cells. Experiments to untangle these differences are fundamental to advancing the understanding of cell biology in AD and elucidating important pathways for therapeutic development. Human-induced pluripotent stem cell models are a valuable platform for such experiments. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Triple-seronegative myasthenia gravis: clinical and epidemiological characteristics.

BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease usually caused by antibodies against the acetylcholine receptor (AChR-Abs), muscle-specific tyrosine kinase (MuSK-Abs), or low-density lipoprotein receptor-related protein 4 (LRP4-Abs). However, there are MG patients who do not have these antibodies and are thus said to have triple-seronegative (triple-SN) MG. OBJECTIVE: This study aims to describe the frequency and clinical and epidemiological characteristics of patients with triple-SN MG. METHODS: This was a retrospective cross-sectional study carried out through the analysis of medical records. Descriptive and analytical statistical analysis was performed comparing subgroups of myasthenic patients, classified according to serological profile. RESULTS: The sample population consisted of 93 MG patients: 85 were positive for antibodies, 80 (86%) with AChR-Abs, 5 (5.4%) with MuSK-Abs, and no MG patients with LRP4-Abs. Eight patients (8.6%) had triple-SN MG; they had a median age at disease onset of 30 years (21-45). Their most common initial symptoms were ptosis, diplopia, and generalized weakness. Most patients presented with mild symptoms at their last visit, reflecting a median MG composite scale score of 4 (0-6), and 75% of patients had an adequate response to treatment. CONCLUSION: Our study showed a low frequency of triple-SN MG in Brazilian MG patients. Triple-SN MG was predominant in females, who presented with ptosis, diplopia, and generalized weakness, and most patients had an adequate response to immunosuppressive treatment. There was no significant difference between triple-SN MG and the other subgroups.

ANTECEDENTES: A Miastenia gravis (MG) é uma desordem autoimune geralmente causada por anticorpos antirreceptores de acetilcolina (anti-RACh), tirosina quinase músculo-específica (anti-MuSK) ou proteína 4 relacionada ao receptor de lipoproteína de baixa densidade (anti-LRP4). No entanto, em uma parcela dos pacientes, nenhum destes três anticorpos pôde ser detectado, sendo estes casos denominados "triplo-soronegativos". OBJETIVO: Descrever a frequência, bem como as características clínicas e epidemiológicas dos pacientes com MG triplo-soronegativa. MéTODOS: Consiste em um estudo transversal e restrospectivo, realizado através da análise de prontuários médicos. Foi realizada análise estatística descritiva e analítica entre os subgrupos de pacientes, classificados de acordo com o perfil sorológico. RESULTADOS: A população consistiu de 93 pacientes com MG: 85 pacientes apresentavam positividade para anticorpos, sendo 80 (86%) com anticorpos anti-RACh, cinco (5,4%) com anti-MuSK, e não foram encontrados pacientes com anti-LRP4. Oito (8,6%) eram pacientes triplo-soronegativos, que apresentaram idade média de início da doença de 30 anos (21-45), e com sintomas iniciais mais comuns de ptose, diplopia e fraqueza generalizada. 75% dos pacientes triplo-soronegativos apresentaram resposta adequada ao tratamento. CONCLUSãO: O estudo demonstrou uma baixa frequência da pacientes com MG triplo-soronegativa na população brasileira. A MG triplo-soronegativa foi predominante nas mulheres, que se apresentaram com ptose, diplopia ou fraqueza generalizada, e a maioria dos pacientes apresentou resposta adequada ao tratamento imunossupressor. Não houve diferença significativa entre a MG triplo-soronegativa e os demais subgrupos.

A familial missense variant in the Alzheimer's disease gene SORL1 impairs its maturation and endosomal sorting.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia.

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

Genetic overlap between ALS and other neurodegenerative or neuromuscular disorders.

OBJECTIVE: In Norway, 89% of patients with Amyotrophic lateral sclerosis (ALS) lacks a genetic diagnose. ALS genes and genes that cause other neuromuscular or neurodegenerative disorders extensively overlap. This population-based study examined whether patients with ALS have a family history of neurological disorders and explored the occurrence of rare genetic variants associated with other neurodegenerative or neuromuscular disorders. METHODS: During a two-year period, blood samples and clinical data from patients with ALS were collected from all 17 neurological departments in Norway. Our genetic analysis involved exome sequencing and bioinformatics filtering of 510 genes associated with neurodegenerative and neuromuscular disorders. The variants were interpreted using genotype-phenotype correlations and bioinformatics tools. RESULTS: A total of 279 patients from a Norwegian population-based ALS cohort participated in this study. Thirty-one percent of the patients had first- or second-degree relatives with other neurodegenerative disorders, most commonly dementia and Parkinson's disease. The genetic analysis identified 20 possible pathogenic variants, in ATL3, AFG3L2, ATP7A, BICD2, HARS1, KIF1A, LRRK2, MSTO1, NEK1, NEFH, and SORL1, in 25 patients. NEK1 risk variants were present in 2.5% of this ALS cohort. Only four of the 25 patients reported relatives with other neurodegenerative or neuromuscular disorders. CONCLUSION: Gene variants known to cause other neurodegenerative or neuromuscular disorders, most frequently in NEK1, were identified in 9% of the patients with ALS. Most of these patients had no family history of other neurodegenerative or neuromuscular disorders. Our findings indicated that AFG3L2, ATP7A, BICD2, KIF1A, and MSTO1 should be further explored as potential ALS-causing genes.

An Additional Lrp4 High Bone Mass Mutation Mitigates the Sost-Knockout Phenotype in Mice by Increasing Bone Remodeling.

Pathogenic variants disrupting the binding between sclerostin (encoded by SOST) and its receptor LRP4 have previously been described to cause sclerosteosis, a rare high bone mass disorder. The sclerostin-LRP4 complex inhibits canonical WNT signaling, a key pathway regulating osteoblastic bone formation and a promising therapeutic target for common bone disorders, such as osteoporosis. In the current study, we crossed mice deficient for Sost (Sost-/-) with our p.Arg1170Gln Lrp4 knock-in (Lrp4KI/KI) mouse model to create double mutant Sost-/-;Lrp4KI/KI mice. We compared the phenotype of Sost-/- mice with that of Sost-/-;Lrp4KI/KI mice, to investigate a possible synergistic effect of the disease-causing p.Arg1170Trp variant in Lrp4 on Sost deficiency. Interestingly, presence of Lrp4KI alleles partially mitigated the Sost-/- phenotype. Cellular and dynamic histomorphometry did not reveal mechanistic insights into the observed phenotypic differences. We therefore determined the molecular effect of the Lrp4KI allele by performing bulk RNA sequencing on Lrp4KI/KI primary osteoblasts. Unexpectedly, mostly genes related to bone resorption or remodeling (Acp5, Rankl, Mmp9) were upregulated in Lrp4KI/KI primary osteoblasts. Verification of these markers in Lrp4KI/KI, Sost-/- and Sost-/-;Lrp4KI/KI mice revealed that sclerostin deficiency counteracts this Lrp4KI/KI effect in Sost-/-;Lrp4KI/KI mice. We therefore hypothesize that models with two inactivating Lrp4KI alleles rather activate bone remodeling, with a net gain in bone mass, whereas sclerostin deficiency has more robust anabolic effects on bone formation. Moreover, these effects of sclerostin and Lrp4 are stronger in female mice, contributing to a more severe phenotype than in males and more detectable phenotypic differences among different genotypes.

A novel homozygous missense variant in LRP4 causing Cenani-Lenz syndactyly syndrome and literature review.

BACKGROUND: Cenani-Lenzsyndactyly syndrome (CLSS; OMIM 212780) is a rare autosomal recessive acral deformity, which is mainly manifested in the fusion of fingers or toes, disordered phalangeal structure, shortening or fusion of the radius and ulna, and renal hypoplasia. CASE PRESENTATION: Our report described an individual with mild phenotypes from China. His parents were not consanguineous. The affected individual was non-dysmorphic. Standard X-ray showed that the both hands have only four metacarpal bones. The distal end of the first metacarpal bone on the right was relatively slender, and the distal phalanx was absent. Multiple phalanges and some soft tissues of both hands were fused. Exome sequencing revealed a novel biallelic c.282C⟩Avariant in low-density lipoprotein receptor-related protein 4 (LRP4; OMIM604270; NM_002334.4) causing p. (Asn94Lys) change in the encoded protein. This variant is predicted to be potentially pathogenic, affecting protein structure and function. CONCLUSION: We report a novel missense variant present in homozygosity in LRP4 to broaden the pathogenic spectrum of LRP4 in syndactyly, and exome sequencing technology is a powerful tool for genetic analysis in prenatal diagnosis and medical research, as a preferred method for the diagnosis of syndactyly and related phenotypes.

LRP4-related signalling pathways and their regulatory role in neurological diseases.

The mechanism of action of low-density lipoprotein receptor related protein 4 (LRP4) is mediated largely via the Agrin-LRP4-MuSK signalling pathway in the nervous system. LRP4 contributes to the development of synapses in the peripheral nervous system (PNS). It interacts with signalling molecules such as the amyloid beta-protein precursor (APP) and the wingless type protein (Wnt). Its mechanisms of action are complex and mediated via interaction between the pre-synaptic motor neuron and post-synaptic muscle cell in the PNS, which enhances the development of the neuromuscular junction (NMJ). LRP4 may function differently in the central nervous system (CNS) than in the PNS, where it regulates ATP and glutamate release via astrocytes. It mayaffect the growth and development of the CNS by controlling the energy metabolism. LRP4 interacts with Agrin to maintain dendrite growth and density in the CNS. The goal of this article is to review the current studies involving relevant LRP4 signaling pathways in the nervous system. The review also discusses the clinical and etiological roles of LRP4 in neurological illnesses, such as myasthenia gravis, Alzheimer's disease and epilepsy. In this review, we provide a theoretical foundation for the pathogenesis and therapeutic application of LRP4 in neurologic diseases.

LRP4 site-specific variants in the third ß-propeller domain causes congenital myasthenic syndrome type 17.

LRP4 is expressed in many organs. It mediates SOST-dependent inhibition of bone formation and acts as an inhibitor of WNT signaling. It is also a postsynaptic end plate cell surface receptor at the neuromuscular junction and is central to its development, maintenance, and function. Pathogenic variants of LRP4 that specifically affect the canonical WNT signaling pathway are known to be associated with Cenani-Lenz syndactyly syndrome or the overlapping condition sclerosteosis. However, site-specific pathogenic variants of LRP4 have been associated with the congenital myasthenic syndrome (CMS) type 17 with no abnormal bone phenotype. Only two studies reported biallelic variants of LRP4 associated with CMS17 that presented during childhood. All three reported variants (NM_002334.4: p.Glu1233Ala, p.Glu1233Lys, or p.Arg1277His) are located within the 3'-edge of the third ß-propeller domain of LRP4. We report on a patient with a biallelic variant of the LRP4 gene presenting with a severe and neonatal lethal phenotype; we also provide a literature review of the previously reported patients. A female neonate, born to healthy consanguineous parents, presented with severe hypotonia, congenital diaphragmatic hernia, pulmonary hypertension, and progressive hypoxemia. Two of her siblings presented with a similar condition in the past, and all three died shortly after birth. Clinical exome sequencing revealed homozygosity for the pathogenic variant NM_002334.4:c.3698A > C (p.[Glu1233Ala]).

Potentially Pathogenic SORL1 Mutations Observed in Autosomal-Dominant Cases of Alzheimer's Disease Do Not Modulate APP Physiopathological Processing.

The SORL1 gene encodes LR11/SorLA, a protein that binds ß-amyloid precursor protein (APP) and drives its intracellular trafficking. SORL1 mutations, occurring frequently in a subset of familial cases of Alzheimer's disease (AD), have been documented, but their pathogenic potential is not yet clear and questions remain concerning their putative influence on the physiopathological processing of APP. We have assessed the influence of two SORL1 mutations that were described as likely disease-causing and that were associated with either benign (SorLA924) or severe (SorLA511) AD phenotypes. We examined the influence of wild-type and mutants SorLA in transiently transfected HEK293 cells expressing either wild-type or Swedish mutated APP on APP expression, secreted Aß and sAPPα levels, intracellular Aß 40 and Aß42 peptides, APP-CTFs (C99 and C83) expressions, α-, ß- and γ-secretases expressions and activities as well as Aß and CTFs-degrading enzymes. These paradigms were studied in control conditions or after pharmacological proteasomal modulation. We also established stably transfected CHO cells expressing wild-type SorLA and established the colocalization of APP and either wild-type or mutant SorLA. SorLA mutations partially disrupt co-localization of wild-type sorLA with APP. Overall, although we mostly confirmed previous data concerning the influence of wild-type SorLA on APP processing, we were unable to evidence significant alterations triggered by our set of SorLA mutants, whatever the cells or pharmacological conditions examined. Our study , however, does not rule out the possibility that other AD-linked SORL1 mutations could indeed affect APP processing, and that pathogenic mutations examined in the present study could interfere with other cellular pathways/triggers in AD.

Pharmacologic enhancement of retromer rescues endosomal pathology induced by defects in the Alzheimer's gene SORL1.

The SORL1 gene (SORLA) is strongly associated with risk of developing Alzheimer's disease (AD). SORLA is a regulator of endosomal trafficking in neurons and interacts with retromer, a complex that is a "master conductor" of endosomal trafficking. Small molecules can increase retromer expression in vitro, enhancing its function. We treated hiPSC-derived cortical neurons that are either fully deficient, haploinsufficient, or that harbor one copy of SORL1 variants linked to AD with TPT-260, a retromer-enhancing molecule. We show significant increases in retromer subunit VPS26B expression. We tested whether endosomal, amyloid, and TAU pathologies were corrected. We observed that the degree of rescue by TPT-260 treatment depended on the number of copies of functional SORL1 and which SORL1 variant was expressed. Using a disease-relevant preclinical model, our work illuminates how the SORL1-retromer pathway can be therapeutically harnessed.

Patient with PSEN1 Glu318Gly and Other Possible Disease Risk Mutations, Diagnosed with Early Onset Alzheimer's Disease.

In this manuscript, we introduced a French EOAD patient in Korea who carried the presenilin-1 (PSEN1) Glu318Gly mutations with four possible risk variants, including sortilin-related receptor 1 (SORL1) Glu270Lys, ATP-binding cassette subfamily A member 7 (ABCA7) Val1946Met, translocase of outer mitochondrial membrane 40 (TOMM40) Arg239Trp, and granulin (GRN) Ala505Gly. The patient started to present memory decline and behavioral dysfunction in his early 60s. His brain imaging presented amyloid deposits by positron emission tomography (PET-CT). The multimer detection system (MDS) screening test for plasma for amyloid oligomers was also positive, which supported the AD diagnosis. It was verified that PSEN1 Glu318Gly itself may not impact amyloid production. However, additional variants were found in other AD and non-AD risk genes, as follows: SORL1 Glu270Lys was suggested as a risk mutation for AD and could increase amyloid peptide production and impair endosome functions. ABCA7 Val1946Met was a novel variant that was predicted to be damaging. The GRN Ala505Gly was a variant with uncertain significance; however, it may reduce the granulin levels in the plasma of dementia patients. Pathway analysis revealed that PSEN1 Glu318Gly may work as a risk factor along with the SORL1 and ABCA7 variants since pathway analysis revealed that PSEN1 could directly interact with them through amyloid-related and lipid metabolism pathways. TOMM40 and PSEN1 could have common mechanisms through mitochondrial dysfunction. It may be possible that PSEN1 Glu318Gly and GRN Ala505Gly would impact disease by impairing immune-related pathways, including microglia and astrocyte development, or NFkB-related pathways. Taken together, the five risk factors may contribute to disease-related pathways, including amyloid and lipid metabolism, or impair immune mechanisms.

LRP10, PGK1 and RPLP0: Best Reference Genes in Periprostatic Adipose Tissue under Obesity and Prostate Cancer Conditions.

Obesity (OB) is a metabolic disorder characterized by adipose tissue dysfunction that has emerged as a health problem of epidemic proportions in recent decades. OB is associated with multiple comorbidities, including some types of cancers. Specifically, prostate cancer (PCa) has been postulated as one of the tumors that could have a causal relationship with OB. Particularly, a specialized adipose tissue (AT) depot known as periprostatic adipose tissue (PPAT) has gained increasing attention over the last few years as it could be a key player in the pathophysiological interaction between PCa and OB. However, to date, no studies have defined the most appropriate internal reference genes (IRGs) to be used in gene expression studies in this AT depot. In this work, two independent cohorts of PPAT samples (n = 20/n = 48) were used to assess the validity of a battery of 15 literature-selected IRGs using two widely used techniques (reverse transcription quantitative PCR [RT-qPCR] and microfluidic-based qPCR array). For this purpose, ΔCt method, GeNorm (v3.5), BestKeeper (v1.0), NormFinder (v.20.0), and RefFinder software were employed to assess the overall trends of our analyses. LRP10, PGK1, and RPLP0 were identified as the best IRGs to be used for gene expression studies in human PPATs, specifically when considering PCa and OB conditions.

Digoxin protects against intervertebral disc degeneration via TNF/NF-κB and LRP4 signaling.

Background: Intervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP). The pathological process of IVDD is associated with inflammatory reactions and extracellular matrix (ECM) disorders. Digoxin is widely used for treating heart failure, and it has been reported to have anti-inflammatory effects. Objective: This study is to investigate the role of digoxin in the pathogenesis of intervertebral disc degeneration as well as the involved molecular mechanism, particularly the potential target protein. Methods: We exploited a rat needle model to investigate digoxin's role in intervertebral disc degeneration in vivo. Safranin O staining was used to measure cartilaginous tissue in the intervertebral disc. The morphological changes of intervertebral discs in animal models were determined by Hematoxylin-Eosin (H&E) staining and the pathological score. Primary nucleus pulposus cells (NP cells) from intervertebral discs of patients and murine were used in the present study. Western-Blotting assay, Real-time PCR assay, immunofluorescence staining, and immunochemistry were used to detect the role of digoxin in anti-TNF-α-induced inflammatory effects in vitro. Transfection of siRNA was used to regulate low-density lipoprotein receptor-related protein 4 (LRP4) expression in NP cells to investigate the potential protein target of digoxin. Results: Digoxin protected against intervertebral disc degeneration in rat needle models. Digoxin was found to exert its disc-protective effects through at least three different pathways by a) suppressing TNF-α-induced inflammation, b) attenuating ECM destruction, c) significantly promoting ECM anabolism. Additionally, LRP4 was found to be the downstream molecule of digoxin in NP cells for anti-inflammation and regulation of ECM metabolism. The knockdown of LRP4 downregulated the protective effect of digoxin in NP cells. Conclusion: These findings suggest that digoxin may be a potential therapeutic agent for intervertebral disc degeneration through anti-catabolism and pro-anabolism. Digoxin might also work as an alternative for other inflammation-related diseases.

Associations of Germline Genetic Variants With Depression and Fatigue Among Hematologic Cancer Patients Treated With Allogeneic Hematopoietic Cell Transplantation.

OBJECTIVE: Depression and fatigue are common among cancer patients and are associated with germline genetic variation. The goal of this pilot study was to examine genetic associations with depression and fatigue in the year after allogeneic hematopoietic cell transplant (HCT). METHODS: Blood was collected from patients and their donors before HCT. Patients completed self-report measures of depression and fatigue before HCT (T1), 90 days post-HCT (T2), and 1 year post-HCT (T3). Of the 384 genetic variants genotyped on a custom Illumina BeadChip microarray, 267 were retained for analysis based on quality control. Main effects of patient and donor variants as well as their interaction were examined using regression analyses. Significant variants were defined as those with a false discovery rate-adjusted p value of <.05. RESULTS: The sample consisted of 59 patient-donor pairs. Mean levels of depression and fatigue did not change significantly over time ( p values of > .41). Increases in depression from T1 to T2 were associated with patient-donor interactions at rs1928040 ( p = 3.0 × 10 -4 ) and rs6311 ( p = 2.0 × 10 -4 ) in HTR2A . Increases in fatigue from T1 to T2 were associated with patient rs689021 in SORL1 ( p = 6.0 × 10 -5 ) and a patient-donor interaction at rs1885884 in HTR2A ( p < 1.0 × 10 -4 ). CONCLUSIONS: Data suggest that variants in genes regulating the serotonergic system ( HTR2A ) and lipid metabolism ( SORL1 ) are associated with changes in depression and fatigue in allogeneic HCT patients, implicating patients' own genetic inheritance as well as that of donors. Additional studies are warranted to confirm these findings.

Thymosin ß4 preserves vascular smooth muscle phenotype in atherosclerosis via regulation of low density lipoprotein related protein 1 (LRP1).

Atherosclerosis is a progressive, degenerative vascular disease and a leading cause of morbidity and mortality. In response to endothelial damage, platelet derived growth factor (PDGF)-BB induced phenotypic modulation of medial smooth muscle cells (VSMCs) promotes atherosclerotic lesion formation and destabilisation of the vessel wall. VSMC sensitivity to PDGF-BB is determined by endocytosis of Low density lipoprotein receptor related protein 1 (LRP1)-PDGFR ß complexes to balance receptor recycling with lysosomal degradation. Consequently, LRP1 is implicated in various arterial diseases. Having identified Tß4 as a regulator of LRP1-mediated endocytosis to protect against aortic aneurysm, we sought to determine whether Tß4 may additionally function to protect against atherosclerosis, by regulating LRP1-mediated growth factor signalling. By single cell transcriptomic analysis, Tmsb4x, encoding Tß4, strongly correlated with contractile gene expression and was significantly down-regulated in cells that adopted a modulated phenotype in atherosclerosis. We assessed susceptibility to atherosclerosis of global Tß4 knockout mice using the ApoE-/- hypercholesterolaemia model. Inflammation, elastin integrity, VSMC phenotype and signalling were analysed in the aortic root and descending aorta. Tß4KO; ApoE-/- mice develop larger atherosclerotic plaques than control mice, with medial layer degeneration characterised by accelerated VSMC phenotypic modulation. Defects in Tß4KO; ApoE-/- mice phenocopied those in VSMC-specific LRP1 nulls and, moreover, were underpinned by hyperactivated LRP1-PDGFRß signalling. We identify an atheroprotective role for endogenous Tß4 in maintaining differentiated VSMC phenotype via LRP1-mediated PDGFRß signalling.

Cell-type-specific regulation of APOE and CLU levels in human neurons by the Alzheimer's disease risk gene SORL1.

SORL1 is implicated in the pathogenesis of Alzheimer's disease (AD) through genetic studies. To interrogate the roles of SORL1 in human brain cells, SORL1-null induced pluripotent stem cells (iPSCs) were differentiated to neuron, astrocyte, microglial, and endothelial cell fates. Loss of SORL1 leads to alterations in both overlapping and distinct pathways across cell types, with the greatest effects in neurons and astrocytes. SORL1 loss induces a neuron-specific reduction in apolipoprotein E (APOE) and clusterin (CLU) and altered lipid profiles. Analyses of iPSCs derived from a large cohort reveal a neuron-specific association between SORL1, APOE, and CLU levels, a finding validated in postmortem brain. Enhancement of retromer-mediated trafficking rescues tau phenotypes observed in SORL1-null neurons but does not rescue APOE levels. Pathway analyses implicate transforming growth factor ß (TGF-ß)/SMAD signaling in SORL1 function, and modulating SMAD signaling in neurons alters APOE RNA levels in a SORL1-dependent manner. Taken together, these data provide a mechanistic link between strong genetic risk factors for AD.