Exerkine GPLD1 bridges liver and brain.

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

Tissue nonspecific alkaline phosphatase promotes calvarial progenitor cell cycle progression and cytokinesis via Erk1,2.

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.

Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aβ plaques, immunostaining revealed that both intracellular Aβ1–42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.

Tissue nonspecific alkaline phosphatase (TNAP) is an ectoenzyme that is known for its anti-inflammatory properties in the immune system. Although TNAP is present in nearly every cell, it is highly abundant in brain endothelial cells (BEC). However, the role of TNAP in the BECs and other cells in the neurovascular unit is poorly understood. Our laboratory has previously identified a role for BEC TNAP in vascular integrity. While the loss of cortical TNAP activity has been observed in ischemic stroke, the functional outcomes that result from the loss of the enzyme are less clear. The objective of this study is to determine how TNAP affects post-stroke functional outcomes. We hypothesized that the loss of endothelial TNAP worsens post-stroke sensorimotor deficits. To test this, we generated mice with a conditional deletion of Alpl , the gene that codes for TNAP, in endothelial cells (VE-cKO mice) and compared the results to their littermate controls ( Alpl fl/fl ). Male and female six month-old mice (n=40) received a photothrombotic ischemic stroke (PTS) or sham surgery followed by daily neurological assessment (Clark’s test) and comprehensive sensorimotor testing, i.e. corner test, rotarod, open field, and gait analysis (DigiGait), over a seven-day period. Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p<0.0001). Daily neurological scoring showed higher (worse) scores in VEcKO PTS-mice compared to littermate controls (p=0.004). Quantification of total spontaneous movement via open field testing showed a significant interaction among stroke, genotype, and time (p=0.014). Gait analysis showed that the post-stroke stance/swing ratio, a measure of stability, differed between the left and right hind paws in VEcKO mice compared to controls: right hind paw- genotype x stroke interaction (p=0.041), and left hind paw- genotype x surgery x sex interaction (p=0.005). Significant interactions between surgery and sex were also observed in the right fore paw for stride (p=0.02), stride frequency (p=0.00026), and stride length (p=0.02). These results demonstrate a novel role for endothelial TNAP in functional recovery post-stroke.

Background and Aims Vascular media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is highly expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether a TNAP inhibitor is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. Method To induce vascular calcification, rats received a diet containing 0.30% warfarin and 0.15% vitamin K1 throughout the entire study and were subjected to the following daily treatments: (i) vehicle (n=10) or (ii) 10 mg/kg/day TNAP-inhibitor (n=10) administered via an intraperitoneal catheter from start of the study until sacrifice at week 7. Calcium, phosphorus and parathyroid hormone (PTH) levels were determined in serum samples as these are important determinants of vascular calcification. As TNAP is also expressed in the liver, serum alanine aminotransferase (ALT) and aspartate (AST) levels were analyzed. At sacrifice, vascular calcification was evaluated by measurement of the total calcium content in the arteries and quantification of the area % calcification on Von Kossa stained sections of the aorta. The mRNA expression of osteo/chondrogenic marker genes (runx2, TNAP, SOX9, collagen 1 and collagen 2) was analyzed in the aorta by qPCR to verify whether vascular smooth muscle cells underwent reprogramming towards bone-like cells. Bone histomorphometry was performed on the left tibia to measure static and dynamic bone parameters as TNAP also regulates physiological bone mineralization. Results No differences in serum calcium, phosphorus and PTH levels was observed between both study groups. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily dosing with the TNAP inhibitor (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84±0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70±0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle condition. The inhibitory effects of SBI-425 on vascular calcification were without altering serum liver markers ALT and AST levels. Furthermore, TNAP-inhibitor SBI-425 did not modulate the mRNA expression of osteo/chondrogenic marker genes runx2, TNAP, SOX9, collagen 1 and 2. Dosing with SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Conclusion Dosing with TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification and this without affecting liver function. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

Tissue-nonspecific alkaline phosphatase is an anti-inflammatory nucleotidase.

The functional co-operativity of tissue-nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 during initiation of skeletal mineralization.

P111 Tenocytes extracted from rotator cuff tendons induce TNAP-dependent mineral deposition and express genes related to a hypertrophic chondrocyte differentiation

Disclosure: P. Pierog: Be Biopharma. M. Brophy: Be Biopharma. W. Wong: Be Biopharma. X. Zhang: Be Biopharma. L. Li: Be Biopharma. J. Hickey: Be Biopharma. A. Lundberg: Be Biopharma. W. Bainter: Be Biopharma. C. Bullock: Be Biopharma. S. Keegan: Be Biopharma. S. Bedard-Shurtleff: Be Biopharma. T. Mullen: Be Biopharma. J. Edelstein: Be Biopharma. S. Bhawsar: Be Biopharma. G. Adekanye: Be Biopharma. E. Liberzon: Be Biopharma. S. Singh: Be Biopharma. M. Musial-Siwek: Be Biopharma. A. Hohmann: Be Biopharma. S. Arlauckas: Be Biopharma. A. Lazorchak: Be Biopharma. H. Liu: Be Biopharma. R. Morgan: Be Biopharma. Hypophosphatasia (HPP) is a rare metabolic disorder that causes skeletal abnormalities, chronic pain, and systemic complications. The condition arises from loss-of-function mutations in the ALPL gene that encodes the tissue-nonspecific alkaline phosphatase (TNSALP) enzyme. Deficient TNSALP activity leads to the accumulation of inorganic pyrophosphate (PPi), an inhibitor of bone mineralization, and pyridoxal 5’-phosphate responsible for disease manifestation. To address limitations of current therapies, we have developed a B cell-based cell therapy, termed BE-102. BE-102 is manufactured from primary human B cells by isolating, activating, and precision engineering with CRISPR/Cas9 followed by AAV and homology directed repair mediated insertion of human ALPL gene into CCR5 locus. Engineered TNSALP-producing cells, have properties of long-lived plasma cells and are expected to reduce elevated levels of substrates PPi and PLP, that accumulate in patients with HPP. BE-102 cells were manufactured from six healthy donors and were characterized for viability (84.9 ± 3.5%), CD38+ expression (93.3 ± 4.3%), ALPL transgene integration (45.1 ± 7.2%) and TNSALP secretion rate (9.5 ± 10.2 ng/mL/hour). In vitro pharmacology results demonstrate that BE-102 secretes active TNSALP (409.7 ± 10.2 µUnits/mL), which is capable of rescuing calcium deposition inhibited by PPi in a cell-based in vitro test system. Having demonstrated the in vitro function of BE-102, in vivo studies (N=3) were conducted in immune-deficient NOG-hIL6 mice. The animals received a single intravenous (IV) injection of 2 x107 viable cells. Human TNSALP was detected in serum at every time point starting at Day 1 and up to 6 months following a single dose of BE-102 using a human specific immunocapture assay. Consistent with BE-102 pharmacology, serum ALP activity showed statistically significant increase in BE-102 treated animals as compared to vehicle-treated animals. The in vivo data confirm long-term engraftment and continuous production of active TNSALP. Consistent with the expected pharmacology of BE-102 and construct design of ALPL transgene, human TNSALP activity was detected at day 90 in the bones of animals treated with BE-102 surrogate construct secreting TNSALP. These data confirm the delivery of functional TNSALP secreted by the surrogate construct to the bone, one of the main HPP disease target organs. Finally, clinical chemistry and hematology results from the end of the studies were all within acceptable ranges. Safety data together with the absence of adverse clinical observations and no unscheduled animal deaths support that BE-102 was well-tolerated in vivo. In summary, pharmacology and safety data establish non-clinical proof of concept that BE-102, has the potential to be a disease-modifying therapy by providing sustained level of active TNSALP following a single IV infusion for patients with HPP. Presentation: Monday, July 14, 2025

Human and mouse alkaline phosphatases (AP) are encoded by a multigene family expressed ubiquitously in multiple tissues. Gene knockout (KO) findings have helped define some of the precise exocytic functions of individual isozymes in bone, teeth, the central nervous system, and in the gut. For instance, deficiency in tissue-nonspecific alkaline phosphatase (TNAP) in mice (Alpl (-/-) mice) and humans leads to hypophosphatasia (HPP), an inborn error of metabolism characterized by epileptic seizures in the most severe cases, caused by abnormal metabolism of pyridoxal-5'-phosphate (the predominant form of vitamin B6) and by hypomineralization of the skeleton and teeth featuring rickets and early loss of teeth in children or osteomalacia and dental problems in adults caused by accumulation of inorganic pyrophosphate (PPi). Enzyme replacement therapy with mineral-targeting TNAP prevented all the manifestations of HPP in mice, and clinical trials with this protein therapeutic are showing promising results in rescuing life-threatening HPP in infants. Conversely, TNAP induction in the vasculature during generalized arterial calcification of infancy (GACI), type II diabetes, obesity, and aging can cause medial vascular calcification. TNAP inhibitors, discussed extensively in this book, are in development to prevent pathological arterial calcification. The brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in fatty acid (FA) absorption, in protecting gut barrier function, and in determining the composition of the gut microbiota via its ability to dephosphorylate lipopolysaccharide (LPS). Knockout mice (Akp3 (-/-)) deficient in duodenal-specific IAP (dIAP) become obese, and develop hyperlipidemia and hepatic steatosis when fed a high-fat diet (HFD). These changes are accompanied by upregulation in the jejunal-ileal expression of the Akp6 IAP isozyme (global IAP, or gIAP) and concomitant upregulation of FAT/CD36, a phosphorylated fatty acid translocase thought to play a role in facilitating the transport of long-chain fatty acids into cells. gIAP, but not dIAP, is able to modulate the phosphorylation status of FAT/CD36. dIAP, even though it is expressed in the duodenum, is shed into the gut lumen and is active in LPS dephosphorylation throughout the gut lumen and in the feces. Akp3 (-/-) mice display gut dysbiosis and are more prone to dextran sodium sulfate-induced colitis than wild-type mice. Of relevance, oral administration of recombinant calf IAP prevents the dysbiosis and protects the gut from chronic colitis. Analogous to the role of IAP in the gut, TNAP expression in the liver may have a proactive role from bacterial endotoxin insult. Finally, more recent studies suggest that neuronal death in Alzheimer's disease may also be associated with TNAP function on certain brain-specific phosphoproteins. This review recounts the established roles of TNAP and IAP and briefly discusses new areas of investigation related to multisystemic functions of these isozymes.

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-β1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5′-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-β1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.

Sepsis is a disorder that targets the microcirculation with significant effects at blood-tissue barriers such as the blood-brain barrier (BBB) and gut-vascular barrier (GVB). Intestinal barrier dysfunction is thought to be one of the most important contributors to multi-organ dysfunction in sepsis. As a common site of infection, the impaired gut allows the dissemination of bacteria, which exacerbates the disease pathophysiology, and can lead to long-term morbidity and mortality. The mechanisms that influence altered barrier permeability in the GVB in sepsis and other inflammatory disorders are not well understood. Tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity, which has been shown to be highly expressed on brain microvascular endothelial cells (BMECs), is a viable therapeutic target. TNAP activity is recognized as generally anti-inflammatory in sepsis-associated acute kidney injury (AKI) through mechanisms not well understood, and its role in health and disease at various tissue barriers has yet to be explored. One critical barrier to studying TNAP has been a lack of specific pharmacological and genetic tools. Therefore, we first sought to examine the effects of pharmacological and genetic manipulation of TNAP in experimental sepsis using novel techniques. Our central hypothesis was that pharmacological or genetic depletion of TNAP enzyme activity would result in exaggerated barrier permeability, increased inflammation, and impaired gut homeostasis in experimental sepsis. Initially, a genetic mouse model with over-expression of TNAP on endothelial cells (i.e., VE-cKO) revealed that TNAP-overexpression resulted in better clinical scores and increased survival at 48-hours post-sepsis. Additionally, mice with genetic deletion of TNAP on endothelial cells (i.e., VE-cKO) were used to determine a role for TNAP in GVB homeostasis. We found that VE-cKO septic mice display increased bacterial burden and increased parameters of gut dysbiosis at 8 days post-CLP, including a decreased Firmicutes:Bacteroidetes (F:B) ratio, decreased relative antimicrobial expression, and, at 3 days post-CLP, decreased levels of short-chain fatty acids (SCFAs). At 24 hours post-CLP, permeability to molecules ranging from 625 Da to 69 kDa is increased in VE-cKO ileum compared to Alplfl/fl littermate controls. The differences in permeability between intestinal segments demonstrates that TNAP’s enzyme activity may impact each region using different mechanisms. Ultimately, our results have shown that endothelial TNAP is protective during sepsis via two potential mechanisms: (1) decreasing bacterial burden and gut dysbiosis by 8 days post-CLP, and (2) decreasing intestinal permeability at 24 hours post-CLP. This work demonstrates a protective role for endothelial TNAP against GVB dysfunction which may be

Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis

Mouse primordial germ cells express tissue non-specific alkaline phosphatase (TNAP) during development, but the widespread expression of another alkaline phosphatase gene in the early embryo limits the potential use of this marker to trace germ cells. To attempt to identify germ cells at all stages during embryonic development and to understand the role of TNAP in germ cell ontogeny, mice carrying a beta geo (lacZ/neor) disrupted allele of the TNAP gene were generated by homologous recombination in embryonic stem cells. Using beta-galactosidase activity, the embryonic pattern of TNAP expression was examined from the blastocyst stage to embryonic day 14. Results indicate that primordial germ cell progenitors do not express TNAP prior to gastrulation although at earlier times TNAP expression is found in an extraembryonic lineage destined to form the chorion. In homozygous mutants, primordial germ cells appear unaffected indicating that TNAP is not essential for their development or migration.