Application of 89Zr-DFO*-immuno-PET to assess improved target engagement of a bispecific anti-amyloid-ß monoclonal antibody.

PURPOSE: The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders. METHODS: Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-ß plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease. RESULTS: A 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice. CONCLUSION: 89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aß plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.

Intra- and inter-tooth variation in strontium isotope ratios from prehistoric seals by laser ablation multi-collector inductively coupled plasma mass spectrometry.

RATIONALE: Strontium isotope ratios (87 Sr/86 Sr) in modern-day marine environments are considered to be homogeneous (~0.7092). However, in the Baltic Sea, the Sr ratios are controlled by mixing seawater and continental drainage from major rivers discharging into the Baltic. This pilot study explores if variations in Sr can be detected in marine mammals from archaeological sites in the Baltic Sea. METHODS: 87 Sr/86 Sr ratios were measured in tooth enamel from three seal species by laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS). The method enables micro-sampling of solid materials. This is the first time that the method has been applied to marine samples from archaeological collections. RESULTS: The analyses showed inter-tooth 87 Sr/86 Sr variation suggesting that different ratios can be detected in different regions of the Baltic Sea. Furthermore, the intra-tooth variation suggests possible different geographic origin or seasonal movement of seals within different regions in the Baltic Sea through their lifetime. CONCLUSIONS: The method was successfully applied to archaeological marine samples showing that: (1) the 87 Sr/86 Sr ratio in marine environments is not uniform, (2) 87 Sr/86 Sr differences might reflect differences in ecology and life history of different seal species, and (3) archaeological mobility studies based on 87 Sr/86 Sr ratios in humans should therefore be evaluated together with diet reconstruction.

Magic angle spinning 31P NMR spectroscopy reveals two essentially identical ionization states for the cardiolipin phosphates in phospholipid liposomes.

Specific membrane lipid composition is crucial for optimized structural and functional organization of biological membranes. Cardiolipin is a unique phospholipid and important component of the inner mitochondrial membrane. It is involved in energy metabolism, inner mitochondrial membrane transport, regulation of multiple metabolic reactions and apoptotic cell death. The physico-chemical properties of cardiolipin have been studied extensively but despite all these efforts there is still lingering controversy regarding the ionization of the two phosphate groups of cardiolipin. Results obtained in the 1990s and early 2000s suggested that cardiolipin has two disparate pKa values where one of the protons was proposed to be stabilized by an intramolecular hydrogen bond. This has led to extensive speculations on the roles of these two putative ionization states of cardiolipin in mitochondria. More recently the notion of two pKa values has been challenged and rejected by several groups. These studies relied on external measurements of proton adsorption or electrophoretic mobility of membranes but did not take into account the low pH phase behavior and chemical stability of cardiolipin. Here we used 31P NMR to show that in the physiologically relevant membrane phospholipid environment, cardiolipin carries two negative charges at physiological pH. We additionally demonstrate the pH dependent phase behavior and chemical stability of cardiolipin containing membranes.

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition.

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L-/-) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L-/- mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Subchronic treatment with phencyclidine in adolescence leads to impaired exploratory behavior in adult rats without altering social interaction or N-methyl-D-aspartate receptor binding levels.

Although both the onset of schizophrenia and human phencyclidine (PCP) abuse typically present within the interval from adolescence to early adulthood, the majority of preclinical research employing the PCP model of schizophrenia has been conducted on neonatal or adult animals. The present study was designed to evaluate the behavioral and neurochemical sequelae of subchronic exposure to PCP in adolescence. Male 35-42-day-old Sprague Dawley rats were subcutaneously administered either saline (10 ml · kg(-1) ) or PCP hydrochloride (10 mg · kg(-1) ) once daily for a period of 14 days (n = 6/group). The animals were allowed to withdraw from treatment for 2 weeks, and their social and exploratory behaviors were subsequently assessed in adulthood by using the social interaction test. To examine the effects of adolescent PCP administration on the regulation of N-methyl-D-aspartate receptors (NMDARs), quantitative autoradiography was performed on brain sections of adult, control and PCP-withdrawn rats by using 20 nM (3) H-MK-801. Prior subchronic exposure to PCP in adolescence had no enduring effects on the reciprocal contact and noncontact social behavior of adult rats. Spontaneous rearing in response to the novel testing arena and time spent investigating its walls and floor were reduced in PCP-withdrawn animals compared with control. The long-term behavioral effects of PCP occurred in the absence of persistent deficits in spontaneous locomotion or self-grooming activity and were not mediated by altered NMDAR density. Our results document differential effects of adolescent PCP administration on the social and exploratory behaviors of adult rats, suggesting that distinct neurobiological mechanisms are involved in mediating these behaviors.

Polarization measurements in high-energy deuteron photodisintegration.

We present measurements of the recoil proton polarization for the d(gamma-->,p-->)n reaction at straight theta(c.m.) = 90 degrees for photon energies up to 2.4 GeV. These are the first data in this reaction for polarization transfer with circularly polarized photons. The induced polarization p(y) vanishes above 1 GeV, contrary to meson-baryon model expectations, in which resonances lead to large polarizations. However, the polarization transfer Cx does not vanish above 1 GeV, inconsistent with hadron helicity conservation. Thus, we show that the scaling behavior observed in the d(gamma,p)n cross sections is not a result of perturbative QCD. These data should provide important tests of new nonperturbative calculations in the intermediate energy regime.