Pantothenate Kinase Activation Restores Brain Coenzyme A in a Mouse Model of Pantothenate Kinase-Associated Neurodegeneration.

Pantothenate kinase-associated neurodegeneration (PKAN) is characterized by a motor disorder with combinations of dystonia, parkinsonism, and spasticity, leading to premature death. PKAN is caused by mutations in the PANK2 gene that result in loss or reduction of PANK2 protein function. PANK2 is one of three kinases that initiate and regulate coenzyme A biosynthesis from vitamin B5, and the ability of BBP-671, an allosteric activator of pantothenate kinases, to enter the brain and elevate coenzyme A was investigated. The metabolic stability, protein binding, and membrane permeability of BBP-671 all suggest that it has the physical properties required to cross the blood-brain barrier. BBP-671 was detected in plasma, liver, cerebrospinal fluid, and brain following oral administration in rodents, demonstrating the ability of BBP-671 to penetrate the brain. The pharmacokinetic and pharmacodynamic properties of orally administered BBP-671 evaluated in cannulated rats showed that coenzyme A (CoA) concentrations were elevated in blood, liver, and brain. BBP-671 elevation of whole-blood acetyl-CoA served as a peripheral pharmacodynamic marker and provided a suitable method to assess target engagement. BBP-671 treatment elevated brain coenzyme A concentrations and improved movement and body weight in a PKAN mouse model. Thus, BBP-671 crosses the blood-brain barrier to correct the brain CoA deficiency in a PKAN mouse model, resulting in improved locomotion and survival and providing a preclinical foundation for the development of BBP-671 as a potential treatment of PKAN. SIGNIFICANCE STATEMENT: The blood-brain barrier represents a major hurdle for drugs targeting brain metabolism. This work describes the pharmacokinetic/pharmacodynamic properties of BBP-671, a pantothenate kinase activator. BBP-671 crosses the blood-brain barrier to correct the neuron-specific coenzyme A (CoA) deficiency and improve motor function in a mouse model of pantothenate kinase-associated neurodegeneration. The central role of CoA and acetyl-CoA in intermediary metabolism suggests that pantothenate kinase activators may be useful in modifying neurological metabolic disorders.

Dermal and oral exposure risks to heavy metals from 3D printing metal-fill thermoplastics.

Manufacturing advancements in polymer printing now allow for the addition of metal additives to thermoplastic feedstock up to 80-90 % by weight and subsequent printing on low-cost desktop 3D printers. Particles associated with metal additives are not chemically bound to the plastic polymer, meaning these particles can potentially migrate and become bioavailable. This study investigated the degree to which two human exposure pathways, oral (ingestion) and dermal (skin contact), are important exposure pathways for metals (copper, chromium, and tin) from metal-fill thermoplastics used in consumer fused filament fabrication (FFF). We found that dermal exposure to copper and bronze filaments presents the highest exposure risk due to chloride (Cl-) in synthetic sweat driving copper (Cu2+) release and dissolution. Chromium and tin were released as micron-sized particles < 24 µm in diameter with low bioaccessibility during simulated oral and dermal exposure scenarios, with potential to undergo dissolution in the gastrointestinal tract based on testing using synthetic stomach fluids. The rate of metal particle release increased by one to two orders of magnitude when thermoplastics were degraded under 1 year of simulated UV weathering. This calls into question the long-term suitability of biodegradable polymers such as PLA for use in metal-fill thermoplastics if they are designed not to be sintered. The greatest exposure risk appears to be from the raw filaments rather than the printed forms, with the former having higher metal release rates in water and synthetic body fluids for all but one filament type. For brittle feedstock that requires greater handling, as metal-fill thermoplastics can be, practices common in metal powder 3D printing such as wearing gloves and washing hands may adequately reduce metal exposure risks.

Patient and caregiver experiences with pantothenate kinase-associated neurodegeneration (PKAN): results from a patient community survey.

BACKGROUND: Pantothenate kinase-associated neurodegeneration (PKAN) is a rare autosomal recessive genetic disorder of PANK2, which enables mitochondrial synthesis of coenzyme A. Its loss causes neurodegeneration with iron accumulation primarily in motor-related brain areas. Symptoms include dystonia, parkinsonism, and other disabilities. PKAN has been categorized as classic PKAN, with an age of onset ≤ 10 years, rapid progression, and early disability or death; and atypical PKAN, with later onset, slower progression, generally milder, and more diverse symptom manifestations. Available treatments are mostly palliative. Information on the lived experience of patients with PKAN and their caregivers or on community-level disease burden is limited. It is necessary to engage patients as partners to expand our understanding and improve clinical outcomes. This patient-oriented research study used multiple-choice and free-form question surveys distributed by patient organizations to collect information on the manifestations and disease burden of PKAN. It also assessed respondents' experiences and preferences with clinical research to inform future clinical trials. RESULTS: The analysis included 166 surveys. Most respondents (87%) were parents of a patient with PKAN and 7% were patients, with 80% from Europe and North America. The study cohort included 85 patients with classic PKAN (mean ± SD age of onset 4.4 ± 2.79 years), 65 with atypical PKAN (13.8 ± 4.79 years), and 16 identified as "not sure". Respondents reported gait disturbances and dystonia most often in both groups, with 44% unable to walk. The classic PKAN group reported more speech, swallowing, and visual difficulties and more severe motor problems than the atypical PKAN group. Dystonia and speech/swallowing difficulties were reported as the most challenging symptoms. Most respondents reported using multiple medications, primarily anticonvulsants and antiparkinsonian drugs, and about half had participated in a clinical research study. Study participants reported the most difficulties with the physical exertion associated with imaging assessments and travel to assessment sites. CONCLUSIONS: The survey results support the dichotomy between classic and atypical PKAN that extends beyond the age of onset. Inclusion of patients as clinical research partners shows promise as a pathway to improving clinical trials and providing more efficacious PKAN therapies.

Clinical and radiological outcomes following surgical hip dislocation for paediatric hip pathologies, a prospective cohort study.

BACKGROUND: Surgical Hip Dislocation (SHD) is a powerful tool in the armamentarium of any surgeon treating conditions affecting the hips of children presenting with sequelae of a number of common conditions including Legg-CalvéPerthes disease (LCPD) and slipped capital femoral epiphysis (SCFE). Risks associated with the procedure are well described. We investigated to assess if SHD is associated with significant surgical risk and if it improved clinical outcomes for patients. METHODS: We conducted a prospective cohort study. We reviewed 18 (11 males and 7 females; mean age 13.7 years (6-17) with symptomatic hip pathology, secondary to femoroacetabular impingement (FAI) between 2017 and 2021. All patients underwent a surgical hip dislocation approach and femoral head-neck osteochondroplasty, Head Split osteotomy or both. Clinical improvement was assessed using the Western Ontario and McMaster Universities Osteoarthritis (WOMAC) index. The minimum follow-up was 6 months (mean, 22 months; range, 6-42 months). RESULTS: WOMAC scores improved at final follow-up from 10 to 3 for pain, 33 to 10 for function, and 4 to 2 for the stiffness subscales. All radiographic measures improved significantly of the postoperative X-rays. No patients developed osteonecrosis, implant failure, deep infection, or nonunion. CONCLUSION: Surgical Hip Dislocation, in the short term, we found improvement in WOMAC scores and radiographic indices with a low complication rate.

Measuring Sulfatase Expression and Invasion in Glioblastoma.

Extracellular sulfatases (SULF1 and SULF2) selectively remove 6-O-sulfate groups (6OS) from heparan sulfate proteoglycans (HSPGs) and by this process control important interactions of HSPGs with extracellular factors including morphogens, growth factors, and extracellular matrix (ECM) components. The expression of SULF1 and SULF2 is dynamically regulated during development and is altered in pathological states such as glioblastoma (GBM), a highly malignant and highly invasive brain cancer. SULF2 protein is increased in an important subset of human GBM and it helps regulate receptor tyrosine kinase (RTK) signaling and tumor growth in a murine model of the disease. By altering ligand binding to HSPGs SULF2 has the potential to modify the extracellular availability of factors important in a number of cell processes including proliferation, chemotaxis, and migration. Diffuse invasion of malignant tumor cells into surrounding healthy brain is a characteristic feature of GBM that makes therapy challenging. Here, we describe methods to assess SULF2 expression in human tumor tissue and cell lines and how to relate this to tumor cell invasion.

Legacy of anthropogenic lead in urban soils: Co-occurrence with metal(loids) and fallout radionuclides, isotopic fingerprinting, and in vitro bioaccessibility.

Anthropogenic lead (Pb) in soils poses risks to human health, particularly to the neuropsychological development of exposed children. Delineating the sources and potential bioavailability of soil Pb, as well as its relationship with other contaminants is critical in mitigating potential human exposure. Here, we present an integrative geochemical analysis of total elemental concentrations, radionuclides of 137Cs and 210Pb, Pb isotopic compositions, and in vitro bioaccessibility of Pb in surface soils sampled from different locations near Durham, North Carolina. Elevated Pb (>400 mg/kg) was commonly observed in soils from urban areas (i.e., near residential house foundation and along urban streets), which co-occurred with other potentially toxic metal(loids) such as Zn, Cd, and Sb. In contrast, soils from city parks and suburban areas had systematically lower concentrations of metal(loids) that were comparable to geological background. The activities of 137Cs and excess 210Pb, coupled with their correlations with Pb and co-occurring metal(loids) were used to indicate the persistence and remobilization of historical atmospherically deposited contaminants. Coupled with total Pb concentrations, the soil Pb isotopic compositions further indicated that house foundation soils had significant input of legacy lead-based paint (mean = 1.1895 and 2.0618 for 206Pb/207Pb and 208Pb/206Pb, respectively), whereas urban streetside soils exhibited a clear mixed origin, dominantly of legacy leaded gasoline (1.2034 and 2.0416) and atmospheric deposition (1.2004-1.2055 and 2.0484-2.0525). The in vitro bioaccessibility of Pb in contaminated urban soils furthermore revealed that more than half of Pb in the contaminated soils was potentially bioavailable, whose Pb isotope ratios were identical to that of bulk soils, demonstrating the utility of using Pb isotopes for tracking human exposure to anthropogenic Pb in soils and house dust. Overall, this study demonstrated a holistic assessment for comprehensively understanding anthropogenic Pb in urban soils, including its co-occurrence with other toxic contaminants, dominant sources, and potential bioavailability upon human exposure.

Urban-Soil Pedogenesis Drives Contrasting Legacies of Lead from Paint and Gasoline in City Soil.

This study analyzed the impact of urban-soil pedogenesis on soil lead (Pb) contamination from paint and gasoline in the historic core of Durham, North Carolina. Total soil Pb in 1000 samples from streetsides, residential properties, and residual upland and floodplains ranged from 6 to 8825 mg/kg (mean = 211 mg/kg), with 50% of samples between 50 and 200 mg/kg soil Pb. The highest Pb concentrations were within 1 m of pre-1978 residential foundations, with concentrations inversely correlated with house age. Streetside soil Pb concentrations were elevated over the geologic background of <30 mg/kg and correlated with traffic flow. Streetside soil Pb concentrations were lower than Durham streetside soils collected in the 1970s, which was attributed to urban pedogenesis, the complex of natural and human processes that change soils over time. Accelerated erosion redistributes legacy Pb and floodplain sampling indicates sedimentation rates of up to 4 mm/year. Mixing and burial of soil with elevated Pb are also lowering soil Pb concentrations over time. These mechanisms are likely of greater significance on streetsides than near foundation soils. The development of an urban-pedogenesis framework can help guide public health approaches to Pb exposure by incorporating pedogenic processes that reduce and dissipate soil Pb contamination.

Heparan Sulfate Synthesized by Ext1 Regulates Receptor Tyrosine Kinase Signaling and Promotes Resistance to EGFR Inhibitors in GBM.

Signaling from multiple receptor tyrosine kinases (RTK) contributes to therapeutic resistance in glioblastoma (GBM). Heparan sulfate (HS), present on cell surfaces and in the extracellular matrix, regulates cell signaling via several mechanisms. To investigate the role for HS in promoting RTK signaling in GBM, we generated neural progenitor cells deficient for HS by knockout of the essential HS-biosynthetic enzyme Ext1, and studied tumor initiation and progression. HS-null cells had decreased proliferation, invasion, and reduced activation of multiple RTKs compared with control. In vivo tumor establishment was significantly decreased, and rate of tumor growth reduced with HS-deficient cells implanted in an HS-poor microenvironment. To investigate if HS regulates RTK activation through platelet-derived growth factor receptor α (PDGFRα) signaling, we removed cell surface HS in patient-derived GBM lines and identified reduced cell surface PDGF-BB ligand. Reduced ligand levels were associated with decreased phosphorylation of PDGFRα, suggesting HS promotes ligand-receptor interaction. Using human GBM tumorspheres and a murine GBM model, we show that ligand-mediated signaling can partially rescue cells from targeted RTK inhibition and that this effect is regulated by HS. Indeed, tumor cells deficient for HS had increased sensitivity to EGFR inhibition in vitro and in vivo. IMPLICATIONS: Our study shows that HS expressed on tumor cells and in the tumor microenvironment regulates ligand-mediated signaling, promoting tumor cell proliferation and invasion, and these factors contribute to decreased tumor cell response to targeted RTK inhibition.

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion.

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults and confers a poor prognosis due, in part, to diffuse invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycans, present on the cell surface and in the extracellular matrix, regulate cell signaling pathways and cell-microenvironment interactions. In GBM, the expression of HS glycosaminoglycans and the enzymes that regulate their function are altered, but the actual HS content and structure are unknown. However, inhibition of HS glycosaminoglycan function is emerging as a promising therapeutic strategy for some cancers. In this study, we use liquid chromatography-mass spectrometry analysis to demonstrate differences in HS disaccharide content and structure across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine GBM with enrichment of mesenchymal and proneural gene expression (mMES and mPN, respectively) markers. In GBM, the heterogeneous HS content and structure across patient-derived tumorsphere lines suggested diverse functions in the GBM tumor microenvironment. In GBM5 and mPN, elevated expression of sulfatase 2 (SULF2), an extracellular enzyme that alters ligand binding to HS, was associated with low trisulfated HS disaccharides, a substrate of SULF2. In contrast, other primary tumorsphere lines had elevated expression of the HS-modifying enzyme heparanase (HPSE). Using gene editing strategies to inhibit HPSE, a role for HPSE in promoting tumor cell adhesion and invasion was identified. These studies characterize the heterogeneity in HS glycosaminoglycan content and structure across GBM and reveal their role in tumor cell invasion.Implications: HS-interacting factors promote GBM invasion and are potential therapeutic targets. Mol Cancer Res; 15(11); 1623-33. ©2017 AACR.

Measuring sulfatase expression and invasion in glioblastoma.

Extracellular sulfatases (SULF1 and SULF2) selectively remove 6-O-sulfate groups from heparan sulfate proteoglycans (HSPGs) and by this process control important interactions of HSPGs with extracellular factors including morphogens, growth factors, and extracellular matrix components. The expression of SULF1 and SULF2 is dynamically regulated during development and is altered in pathological states such as glioblastoma (GBM), a highly malignant and highly invasive brain cancer. SULF2 protein is increased in an important subset of human GBM and it helps regulate receptor tyrosine kinase signaling and tumor growth in a murine model of the disease. By altering ligand binding to HSPGs, SULF2 has the potential to modify the extracellular availability of factors important in a number of cell processes including proliferation, chemotaxis, and migration. Diffuse invasion of malignant tumor cells into surrounding healthy brain is a characteristic feature of GBM that makes therapy challenging. Here, we describe methods to assess SULF2 expression in human tumor tissue and cell lines and how to relate this to tumor cell invasion.

Matrix regulators in neural stem cell functions.

BACKGROUND: Neural stem/progenitor cells (NSPCs) reside within a complex and dynamic extracellular microenvironment, or niche. This niche regulates fundamental aspects of their behavior during normal neural development and repair. Precise yet dynamic regulation of NSPC self-renewal, migration, and differentiation is critical and must persist over the life of an organism. SCOPE OF REVIEW: In this review, we summarize some of the major components of the NSPC niche and provide examples of how cues from the extracellular matrix regulate NSPC behaviors. We use proteoglycans to illustrate the many diverse roles of the niche in providing temporal and spatial regulation of cellular behavior. MAJOR CONCLUSIONS: The NSPC niche is comprised of multiple components that include; soluble ligands, such as growth factors, morphogens, chemokines, and neurotransmitters, the extracellular matrix, and cellular components. As illustrated by proteoglycans, a major component of the extracellular matrix, the NSPC, niche provides temporal and spatial regulation of NSPC behaviors. GENERAL SIGNIFICANCE: The factors that control NSPC behavior are vital to understand as we attempt to modulate normal neural development and repair. Furthermore, an improved understanding of how these factors regulate cell proliferation, migration, and differentiation, crucial for malignancy, may reveal novel anti-tumor strategies. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Proteoglycans and their roles in brain cancer.

Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour-microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets.

Activated leukocyte cell adhesion molecule modulates neurotrophin signaling.

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) have been shown to modulate growth factor signaling and follow complex trafficking pathways in neurons. Similarly, several growth factors, including members of the neurotrophin family, undergo axonal retrograde transport that is required to elicit their full signaling potential in neurons. We sought to determine whether IgCAMs that enter the axonal retrograde transport route co-operate with neurotrophin signaling. We identified activated leukocyte cell adhesion molecule (ALCAM), a protein involved in axon pathfinding and development of the neuromuscular junction, to be associated with an axonal endocytic compartment that contains neurotrophins and their receptors. Although ALCAM enters carriers that are transported bidirectionally in motor neuron axons, it is predominantly co-transported with the neurotrophin receptor p75(NTR) toward the cell body. ALCAM was found to specifically potentiate nerve growth factor (NGF)-induced differentiation and signaling. The extracellular domain of ALCAM is both necessary and sufficient to potentiate NGF-induced neurite outgrowth, and its homodimerization is required for this novel role. Our findings indicate that ALCAM synergizes with NGF to induce neuronal differentiation, raising the possibility that it functions not only as an adhesion molecule but also in the modulation of growth factor signaling in the nervous system.

The effect of N-acetyl-aspartyl-glutamate and N-acetyl-aspartate on white matter oligodendrocytes.

Elevations of the levels of N-acetyl-aspartyl-glutamate (NAAG) and N-acetyl-aspartate (NAA) are associated with myelin loss in the leucodystrophies Canavan's disease and Pelizaeus-Merzbacher-like disease. NAAG and NAA can activate and antagonize neuronal N-methyl-D-aspartate (NMDA) receptors, and also act on group II metabotropic glutamate receptors. Oligodendrocytes and their precursors have recently been shown to express NMDA receptors, and activation of these receptors in ischaemia leads to the death of oligodendrocyte precursors and the loss of myelin. This raises the possibility that the failure to develop myelin, or demyelination, occurring in the leucodystrophies could reflect an action of NAAG or NAA on oligodendrocyte NMDA receptors. However, since the putative subunit composition of NMDA receptors on oligodendrocytes differs from that of neuronal NMDA receptors, the effects of NAAG and NAA on them are unknown. We show that NAAG, but not NAA, evokes an inward membrane current in cerebellar white matter oligodendrocytes, which is reduced by NMDA receptor block (but not by block of metabotropic glutamate receptors). The size of the current evoked by NAAG, relative to that evoked by NMDA, was much smaller in oligodendrocytes than in neurons, and NAAG induced a rise in [Ca(2+)](i) in neurons but not in oligodendrocytes. These differences in the effect of NAAG on oligodendrocytes and neurons may reflect the aforementioned difference in receptor subunit composition. In addition, as a major part of the response in oligodendrocytes was blocked by tetrodotoxin (TTX), much of the NAAG-evoked current in oligodendrocytes is a secondary consequence of activating neuronal NMDA receptors. Six hours exposure to 1 mM NAAG did not lead to the death of cells in the white matter. We conclude that an action of NAAG on oligodendrocyte NMDA receptors is unlikely to be a major contributor to white matter damage in the leucodystrophies.

PDGFRA/NG2 glia generate myelinating oligodendrocytes and piriform projection neurons in adult mice.

Platelet-derived growth factor alpha receptor (PDGFRA)/NG2-expressing glia are distributed throughout the adult CNS. They are descended from oligodendrocyte precursors (OLPs) in the perinatal CNS, but it is not clear whether they continue to generate myelinating oligodendrocytes or other differentiated cells during normal adult life. We followed the fates of adult OLPs in Pdgfra-creER(T2)/Rosa26-YFP double-transgenic mice and found that they generated many myelinating oligodendrocytes during adulthood; >20% of all oligodendrocytes in the adult mouse corpus callosum were generated after 7 weeks of age, raising questions about the function of the late-myelinating axons. OLPs also produced some myelinating cells in the cortex, but the majority of adult-born cortical cells did not appear to myelinate. We found no evidence for astrocyte production in gray or white matter. However, small numbers of projection neurons were generated in the forebrain, especially in the piriform cortex, which is the main target of the olfactory bulb.

Atrophy and degeneration in sciatic nerve of presymptomatic mice carrying the Huntington's disease mutation.

Huntington's disease (HD) is a progressive neurological disorder characterised by motor impairments caused by degeneration in the striatum. The mechanism by which the HD mutation leads to the neurodegenerative pathology of HD is still unknown. Recently it was shown that, in HD patients, early pathological changes in white matter precede selective cell death in the striatum. We wondered whether axonal pathology is also an early pathological feature in a transgenic mouse model carrying the HD mutation (R/2 line). R6/2 mice show brain atrophy, a progressive neurological deterioration and skeletal muscle atrophy that resemble those seen in HD patients. However, there is very little neuronal cell loss seen in these animals, even when they show severe symptoms. Here we used sciatic nerve to look for evidence of early neurodegenerative changes in axons of the R6/2 mouse at an ultrastructural level. We observed ultrastructural changes that preferentially affected large myelinated fibres of the sciatic nerve in 10-week-old asymptomatic R6/2 mice. The changes included a significant decrease in the axoplasm diameter of myelinated neurons and an increase in the number of degenerating myelinated fibres compared to age-matched wild type littermates. Myelin thickness and unmyelinated fibre diameter were not affected. The abnormalities described here precede the appearance of overt motor symptoms in the R6/2 mouse and occur in parallel with pathophysiological changes at the neuromuscular junction. We suggest that degenerative changes in axons are likely to contribute to the early pathological phenotype in HD, even in the absence of frank neuronal cell loss.