The NLRP3 inflammasome modulates tau pathology and neurodegeneration in a tauopathy model.

An active role of neuroinflammation and the NLRP3 inflammasome in Alzheimer's disease and related tauopathies is increasingly identified, supporting NLRP3 as an interesting therapeutic target. However, its effect on tau-associated neurodegeneration, a key-process in tauopathies, remains unknown. While tau pathology and neurodegeneration are closely correlated, different tau forms may act as culprits in both characteristics and NLRP3-dependent microglial processes may differently affect both processes, indicating the need to study the role of NLRP3 in both processes concomitantly. To study the role of NLRP3 on tau pathology, prion-like propagation and tau-associated neurodegeneration we generated crosses of NLRP3 deficient mice with tauP301S (PS19) transgenic mice. In this model we studied non-seeded tau pathology and hippocampal atrophy, reminiscent characteristics of tauopathies. Tau pathology in hippocampus and cortex was significantly decreased in tau.NLRP3-/- versus tau.NLRP3+/+ mice. Importantly, tau.NLRP3-/- mice also displayed significantly decreased hippocampal atrophy, indicating a role of NLRP3 in neurodegeneration. We furthermore assessed the effect of NLRP3 deficiency on tau propagation and associated hippocampal atrophy. NLRP3 deficiency significantly decreased prion-like seeding and propagation of tau pathology, reflected in decreased tau pathology in ipsi- and contralateral hippocampus and cortex in tau.NLRP3-/- following tau seeding. Most importantly, hippocampal atrophy was significantly less in tau-seeded tau.NLRP3-/- mice at 8 months. We here demonstrate for the first time that NLRP3 activation affects tau-associated neurodegeneration and seeded and non-seeded tau pathology, hence affecting key molecular processes in tauopathies. Our data thereby provide key-information in the validation of NLRP3 inflammasome as therapeutic target for AD and related tauopathies.

Passive immunotherapy with a novel antibody against 3pE-modified Aß demonstrates potential for enhanced efficacy and favorable safety in combination with BACE inhibitor treatment in plaque-depositing mice.

The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.

SliceMap: an algorithm for automated brain region annotation.

Summary: Many neurodegenerative disorders, such as Alzheimer's Disease, pertain to or spread from specific sites of the brain. Hence, accurate disease staging or therapy assessment in transgenic model mice demands automated analysis of selected brain regions. To address this need, we have developed an algorithm, termed SliceMap, that enables contextual quantification by mapping anatomical information onto microtome-cut brain slices. For every newly acquired high-resolution image of a brain slice, the algorithm performs a coarse congealing-based registration to a library of pre-annotated reference slices. A subset of optimally matching reference slices is then used for refined, elastic registration. Morphotextural metrics are used to measure registration performance and to automatically detect poorly cut slices. We have implemented our method as a plugin for FIJI image analysis freeware, and we have used it to regionally quantify tau pathology in brain slices from a tauopathy (P301S) mouse model. By enabling region-based quantification, our method contributes to a more accurate assessment of neurodegenerative disease development. Availability and implementation: The method is available as a plugin for FIJI from https://github.com/mbarbie1/SliceMap/, along with an example dataset and user instructions. Contact: winnok.devos@uantwerpen.be. Supplementary information: Supplementary data are available at Bioinformatics online.

Systemic immune-checkpoint blockade with anti-PD1 antibodies does not alter cerebral amyloid-ß burden in several amyloid transgenic mouse models.

Chronic inflammation represents a central component in the pathogenesis of Alzheimer's disease (AD). Recent work suggests that breaking immune tolerance by Programmed cell Death-1 (PD1) checkpoint inhibition produces an IFN-γ-dependent systemic immune response, with infiltration of the brain by peripheral myeloid cells and neuropathological as well as functional improvements even in mice with advanced amyloid pathology (Baruch et al., (): Nature Medicine, 22:135-137). Immune checkpoint inhibition was therefore suggested as potential treatment for neurodegenerative disorders when activation of the immune system is appropriate. Because a xenogeneic rat antibody (mAb) was used in the study, whether the effect was specific to PD1 target engagement was uncertain. In the present study we examined whether PD1 immunotherapy can lower amyloid-ß pathology in a range of different amyloid transgenic models performed at three pharmaceutical companies with the exact same anti-PD1 isotype and two mouse chimeric variants. Although PD1 immunotherapy stimulated systemic activation of the peripheral immune system, monocyte-derived macrophage infiltration into the brain was not detected, and progression of brain amyloid pathology was not altered. Similar negative results of the effect of PD1 immunotherapy on amyloid brain pathology were obtained in two additional models in two separate institutions. These results show that inhibition of PD1 checkpoint signaling by itself is not sufficient to reduce amyloid pathology and that additional factors might have contributed to previously published results (Baruch et al., (): Nature Medicine, 22:135-137). Until such factors are elucidated, animal model data do not support further evaluation of PD1 checkpoint inhibition as a therapeutic modality for Alzheimer's disease.

Comparison of New Tau PET-Tracer Candidates With [18F]T808 and [18F]T807.

Early clinical results of two tau tracers, [(18)F]T808 and [(18)F]T807, have recently been reported. In the present study, the biodistribution, radiometabolite quantification, and competition-binding studies were performed in order to acquire comparative preclinical data as well as to establish the value of T808 and T807 as benchmark compounds for assessment of binding affinities of eight new/other tau tracers. Biodistribution studies in mice showed high brain uptake and fast washout.In vivoradiometabolite analysis using high-performance liquid chromatography showed the presence of polar radiometabolites in plasma and brain. No specific binding of [(18)F]T808 was found in transgenic mice expressing mutant human P301L tau. In semiquantitative autoradiography studies on human Alzheimer disease slices, we observed more than 50% tau selective blocking of [(18)F]T808 in the presence of 1 µmol/L of the novel ligands. This study provides a straightforward comparison of the binding affinity and selectivity for tau of the reported radiolabeled tracers BF-158, BF-170, THK5105, lansoprazole, astemizole, and novel tau positron emission tomography ligands against T807 and T808. Therefore, these data are helpful to identify structural requirements for selective interaction with tau and to compare the performance of new highly selective and specific radiolabeled tau tracers.

Intracerebral injection of preformed synthetic tau fibrils initiates widespread tauopathy and neuronal loss in the brains of tau transgenic mice.

Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.

Structural basis for the oligomerization-facilitated NLRP3 activation.

The NACHT-, leucine-rich-repeat-, and pyrin domain-containing protein 3 (NLRP3) is a critical intracellular inflammasome sensor and an important clinical target against inflammation-driven human diseases. Recent studies have elucidated its transition from a closed cage to an activated disk-like inflammasome, but the intermediate activation mechanism remains elusive. Here we report the cryo-electron microscopy structure of NLRP3, which forms an open octamer and undergoes a ~ 90° hinge rotation at the NACHT domain. Mutations on open octamer's interfaces reduce IL-1ß signaling, highlighting its essential role in NLRP3 activation/inflammasome assembly. The centrosomal NIMA-related kinase 7 (NEK7) disrupts large NLRP3 oligomers and forms NEK7/NLRP3 monomers/dimers which is a critical step preceding the assembly of the disk-like inflammasome. These data demonstrate an oligomeric cooperative activation of NLRP3 and provide insight into its inflammasome assembly mechanism.

Inflammasome signaling is dispensable for ß-amyloid-induced neuropathology in preclinical models of Alzheimer's disease.

Background: Alzheimer's disease (AD) is the most common neurodegenerative disorder affecting memory and cognition. The disease is accompanied by an abnormal deposition of ß-amyloid plaques in the brain that contributes to neurodegeneration and is known to induce glial inflammation. Studies in the APP/PS1 mouse model of ß-amyloid-induced neuropathology have suggested a role for inflammasome activation in ß-amyloid-induced neuroinflammation and neuropathology. Methods: Here, we evaluated the in vivo role of microglia-selective and full body inflammasome signalling in several mouse models of ß-amyloid-induced AD neuropathology. Results: Microglia-specific deletion of the inflammasome regulator A20 and inflammasome effector protease caspase-1 in the AppNL-G-F and APP/PS1 models failed to identify a prominent role for microglial inflammasome signalling in ß-amyloid-induced neuropathology. Moreover, global inflammasome inactivation through respectively full body deletion of caspases 1 and 11 in AppNL-G-F mice and Nlrp3 deletion in APP/PS1 mice also failed to modulate amyloid pathology and disease progression. In agreement, single-cell RNA sequencing did not reveal an important role for Nlrp3 signalling in driving microglial activation and the transition into disease-associated states, both during homeostasis and upon amyloid pathology. Conclusion: Collectively, these results question a generalizable role for inflammasome activation in preclinical amyloid-only models of neuroinflammation.

Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.

Although peroxisome biogenesis and ß-oxidation disorders are well known for their neurodevelopmental defects, patients with these disorders are increasingly diagnosed with neurodegenerative pathologies. In order to investigate the cellular mechanisms of neurodegeneration in these patients, we developed a mouse model lacking multifunctional protein 2 (MFP2, also called D-bifunctional protein), a central enzyme of peroxisomal ß-oxidation, in all neural cells (Nestin-Mfp2(-/-)) or in oligodendrocytes (Cnp-Mfp2(-/-)) and compared these models with an already established general Mfp2 knockout. Nestin-Mfp2 but not Cnp-Mfp2 knockout mice develop motor disabilities and ataxia, similar to the general mutant. Deterioration of motor performance correlates with the demise of Purkinje cell axons in the cerebellum, which precedes loss of Purkinje cells and cerebellar atrophy. This closely mimics spinocerebellar ataxias of patients affected with mild peroxisome ß-oxidation disorders. However, general knockouts have a much shorter life span than Nestin-Mfp2 knockouts which is paralleled by a disparity in activation of the innate immune system. Whereas in general mutants a strong and chronic proinflammatory reaction proceeds throughout the brain, elimination of MFP2 from neural cells results in minor neuroinflammation. Neither the extent of the inflammatory reaction nor the cerebellar degeneration could be correlated with levels of very long chain fatty acids, substrates of peroxisomal ß-oxidation. In conclusion, MFP2 has multiple tasks in the adult brain, including the maintenance of Purkinje cells and the prevention of neuroinflammation but this is not mediated by its activity in oligodendrocytes nor by its role in very long chain fatty acid degradation.

Development and Characterization of Mouse-Specific Anti-Tau Monoclonal Antibodies: Relevance for Analysis of Murine Tau in Cerebrospinal Fluid.

BACKGROUND: Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. OBJECTIVE: We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. METHODS: Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. RESULTS: Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. CONCLUSION: The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.

Peroxisome deficiency but not the defect in ether lipid synthesis causes activation of the innate immune system and axonal loss in the central nervous system.

BACKGROUND: Mice with peroxisome deficiency in neural cells (Nestin-Pex5-/-) develop a neurodegenerative phenotype leading to motor and cognitive disabilities and early death. Major pathologies at the end stage of disease include severe demyelination, axonal degeneration and neuroinflammation. We now investigated the onset and progression of these pathological processes, and their potential interrelationship. In addition, the putative role of oxidative stress, the impact of plasmalogen depletion on the neurodegenerative phenotype, and the consequences of peroxisome elimination in the postnatal period were studied. METHODS: Immunohistochemistry in association with gene expression analysis was performed on Nestin-Pex5-/- mice to document demyelination, axonal damage and neuroinflammation. Also Gnpat-/- mice, with selective plasmalogen deficiency and CMV-Tx-Pex5-/- mice, with tamoxifen induced generalized loss of peroxisomes were analysed. RESULTS: Activation of the innate immune system is a very early event in the pathological process in Nestin-Pex5-/- mice which evolves in chronic neuroinflammation. The complement factor C1q, one of the earliest up regulated transcripts, was expressed on neurons and oligodendrocytes but not on microglia. Transcripts of other pro- and anti-inflammatory genes and markers of phagocytotic activity were already significantly induced before detecting pathologies with immunofluorescent staining. Demyelination, macrophage activity and axonal loss co-occurred throughout the brain. As in patients with mild peroxisome biogenesis disorders who develop regressive changes, demyelination in cerebellum and brain stem preceded major myelin loss in corpus callosum of both Nestin-Pex5-/- and CMV-Tx-Pex5-/- mice. These lesions were not accompanied by generalized oxidative stress throughout the brain. Although Gnpat-/- mice displayed dysmyelination and Purkinje cell axon damage in cerebellum, confirming previous observations, no signs of inflammation or demyelination aggravating with age were observed. CONCLUSIONS: Peroxisome inactivity triggers a fast neuroinflammatory reaction, which is not solely due to the depletion of plasmalogens. In association with myelin abnormalities this causes axon damage and loss.

Peroxisome deficient aP2-Pex5 knockout mice display impaired white adipocyte and muscle function concomitant with reduced adrenergic tone.

Peroxisomes are essential for intermediary lipid metabolism, but the role of these organelles has been primarily studied in the liver. We recently generated aP2-Pex5 conditional knockout mice that due to the nonselectivity of the aP2 promoter, not only had dysfunctional peroxisomes in the adipose tissue but also in the central and peripheral nervous system, besides some other tissues. Peroxisomes were however intact in the liver, heart, pancreas and muscle. Surprisingly, these mice not only showed dysfunctional white adipose tissue with increased fat mass and reduced lipolysis but also the skeletal muscle was affected including impaired shivering thermogenesis, reduced motor performance and increased insulin resistance. Non-shivering thermogenesis by brown adipose tissue was not altered. Strongly reduced levels of plasma adrenaline and to a lesser extent noradrenaline, impaired expression of catecholamine synthesizing enzymes in the adrenal medulla and reversal of all pathologies after administration of the ß-agonist isoproterenol indicated that ß-adrenergic signaling was reduced. Based on normal white adipose and muscle function in Nestin-Pex5 and Wnt-Pex5 knockout mice respectively, it is unlikely that peroxisome absence from the central and peripheral nervous system caused the phenotype. We conclude that peroxisomal metabolism is necessary to maintain the adrenergic tone in mice, which in turn determines metabolic homeostasis.

CSF1R inhibition rescues tau pathology and neurodegeneration in an A/T/N model with combined AD pathologies, while preserving plaque associated microglia.

Alzheimer's disease (AD) is characterized by a sequential progression of amyloid plaques (A), neurofibrillary tangles (T) and neurodegeneration (N), constituting ATN pathology. While microglia are considered key contributors to AD pathogenesis, their contribution in the combined presence of ATN pathologies remains incompletely understood. As sensors of the brain microenvironment, microglial phenotypes and contributions are importantly defined by the pathologies in the brain, indicating the need for their analysis in preclinical models that recapitulate combined ATN pathologies, besides their role in A and T models only. Here, we report a new tau-seed model in which amyloid pathology facilitates bilateral tau propagation associated with brain atrophy, thereby recapitulating robust ATN pathology. Single-cell RNA sequencing revealed that ATN pathology exacerbated microglial activation towards disease-associated microglia states, with a significant upregulation of Apoe as compared to amyloid-only models (A). Importantly, Colony-Stimulating Factor 1 Receptor inhibition preferentially eliminated non-plaque-associated versus plaque associated microglia. The preferential depletion of non-plaque-associated microglia significantly attenuated tau pathology and neuronal atrophy, indicating their detrimental role during ATN progression. Together, our data reveal the intricacies of microglial activation and their contributions to pathology in a model that recapitulates the combined ATN pathologies of AD. Our data may provide a basis for microglia-targeting therapies selectively targeting detrimental microglial populations, while conserving protective populations.

Time Trends of Cerebrospinal Fluid Biomarkers of Neurodegeneration in Idiopathic Normal Pressure Hydrocephalus.

BACKGROUND: Longitudinal changes in cerebrospinal fluid (CSF) biomarkers are seldom studied. Furthermore, data on biomarker gradient between lumbar (L-) and ventricular (V-) compartments seems to be discordant. OBJECTIVE: To examine alteration of CSF biomarkers reflecting Alzheimer's disease (AD)-related amyloid-ß (Aß) aggregation, tau pathology, neurodegeneration, and early synaptic degeneration by CSF shunt surgery in idiopathic normal pressure hydrocephalus (iNPH) in relation to AD-related changes in brain biopsy. In addition, biomarker levels in L- and V-CSF were compared. METHODS: L-CSF was collected prior to shunt placement and, together with V-CSF, 3-73 months after surgery. Thereafter, additional CSF sampling took place at 3, 6, and 18 months after the baseline sample from 26 iNPH patients with confirmed Aß plaques in frontal cortical brain biopsy and 13 iNPH patients without Aß pathology. CSF Amyloid-ß42 (Aß42), total tau (T-tau), phosphorylated tau (P-tau181), neurofilament light (NFL), and neurogranin (NRGN) were analyzed with customized ELISAs. RESULTS: All biomarkers but Aß42 increased notably by 140-810% in L-CSF after CSF diversion and then stabilized. Aß42 instead showed divergent longitudinal decrease between Aß-positive and -negative patients in L-CSF, and thereafter increase in Aß-negative iNPH patients in both L- and V-CSF. All five biomarkers correlated highly between V-CSF and L-CSF (Aß42 R = 0.87, T-tau R = 0.83, P-tau R = 0.92, NFL R = 0.94, NRGN R = 0.9; all p < 0.0001) but were systematically lower in V-CSF (Aß42 14 %, T-tau 22%, P-tau 20%, NFL 32%, NRGN 19%). With APOE genotype-grouping, only Aß42 showed higher concentration in non-carriers of allele ɛ4. CONCLUSION: Longitudinal follow up shows that after an initial post-surgery increase, T-tau, P-tau, and NRGN are stable in iNPH patients regardless of brain biopsy Aß pathology, while NFL normalized toward its pre-shunt levels. Aß42 as biomarker seems to be the least affected by the surgical procedure or shunt and may be the best predictor of AD risk in iNPH patients. All biomarker concentrations were lower in V- than L-CSF yet showing strong correlations.

Axonal integrity in the absence of functional peroxisomes from projection neurons and astrocytes.

Ablation of functional peroxisomes from all neural cells in Nestin-Pex5 knockout mice caused remarkable neurological abnormalities including motoric and cognitive malfunctioning accompanied by demyelination, axonal degeneration, and gliosis. An oligodendrocyte selective Cnp-Pex5 knockout mouse model shows a similar pathology, but with later onset and slower progression. Until now, the link between these neurological anomalies and the known metabolic alterations, namely the accumulation of very long-chain fatty acids (VLCFA) and reduction of plasmalogens, has not been established. We now focused on the role of peroxisomes in neurons and astrocytes. A neuron-specific peroxisome knockout model, NEX-Pex5, showed neither microscopic nor metabolic abnormalities indicating that the lack of functional peroxisomes within neurons does not cause axonal damage. Axonal integrity and normal behavior was also preserved when peroxisomes were deleted from astrocytes in GFAP-Pex5(-/-) mice. Nevertheless, peroxisomal metabolites were dysregulated in brain including a marked accumulation of VLCFA and a slight reduction in plasmalogens. Interestingly, despite minor targeting of oligodendrocytes in GFAP-Pex5(-/-) mice, these metabolic perturbations were also present in isolated myelin indicating that peroxisomal metabolites are shuttled between different brain cell types. We conclude that absence of peroxisomal metabolism in neurons and astrocytes does not provoke the neurodegenerative phenotype observed after deleting peroxisomes from oligodendrocytes. Lack of peroxisomal metabolism in astrocytes causes increased VLCFA levels in myelin, but this has no major impact on neurological functioning.

Combined deficiency of peroxisomal beta-oxidation and ether lipid synthesis in mice causes only minor cortical neuronal migration defects but severe hypotonia.

The metabolic factors causing cortical neuronal migration defects, hypotonia and malformation of cerebellum in patients and mice with severe peroxisome biogenesis disorders are still not identified. In the present investigation, we tested the hypothesis that the combined inactivity of peroxisomal beta-oxidation and ether lipid biosynthesis could be at the origin of these pathologies. Double MFP2/DAPAT knockout mice were generated and their postnatal phenotypes were compared with single knockouts and control mice. Cortical neuronal migration was not affected in DAPAT knockouts and only mildly in double MFP2/DAPAT knockout mice. The latter mice were severely hypotonic and usually died in the postnatal period. Both DAPAT and MFP2 single knockout mice exhibited delays in the formation of cerebellar folia. We conclude that the combined defect of peroxisomal beta-oxidation and ether lipid synthesis does not solely account for the typical cortical neuronal migration defect of mice with peroxisome biogenesis disorders but contributes to their hypotonia.

Direct nose to brain delivery of small molecules: critical analysis of data from a standardized in vivo screening model in rats.

The blood-brain barrier (BBB) is often a limiting factor for getting drugs in the brain. Bypassing the BBB by intranasal (IN), or also called nose to brain (NTB), route is an interesting and frequently investigated concept for brain drug delivery. However, despite the body of evidence for IN drug delivery in literature over the last decades, reproducibility and interpretation of animal data remain challenging. The objective of this project was to assess the feasibility and value of a standardized IN screening model in rats for the evaluation of direct brain delivery. A chemically diverse set of commercial and internal small molecules were tested in the in vivo model with different doses and/or formulations. Data were analyzed using different ways of ratio calculations: blood concentration at time of sacrifice, total exposure in blood (area under the curve, AUC) and the brain or olfactory bulb concentrations. The IN route was compared to another parenteral route to decide if there is potential direct brain transport. The results show that blood and tissue concentrations and ratios are highly variable and not always reproducible. Potential direct brain delivery was concluded for some compounds, however, sometimes depending on the analysis: using blood levels at sacrifice or AUC could lead to different conclusions. We conclude that a screening model for the evaluation of direct brain transport of small molecules is very difficult to achieve and a conclusion based on a limited number of animals with this variability is questionable.

Discovery and Functional Characterization of hPT3, a Humanized Anti-Phospho Tau Selective Monoclonal Antibody.

BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.

Absence of functional peroxisomes from mouse CNS causes dysmyelination and axon degeneration.

Peroxisomal metabolism is essential for normal brain development both in men and in mice. Using conditional knock-out mice, we recently showed that peroxisome deficiency in liver has a severe and persistent impact on the formation of cortex and cerebellum, whereas absence of functional peroxisomes from the CNS only causes developmental delays without obvious alteration of brain architecture. We now report that a substantial fraction of the latter Nes-Pex5 knock-out mice survive into adulthood but develop progressive motoric and coordination problems, impaired exploration, and a deficit in cognition and die before the age of 6 months. Histopathologically, both the white and gray matter of the CNS displayed a region-specific accumulation of neutral lipids, astrogliosis and microgliosis, upregulation of catalase, and scattered cell death. Nes-Pex5 knock-out mice featured a dramatic reduction of myelin staining in corpus callosum, whereas cerebellum and other white matter tracts were less affected or unchanged. This was accompanied by a depletion of alkenylphospholipids in myelin and differentially reduced immunoreactivity of myelin proteins. EM analysis revealed that myelin wrappings around axons did still form, but they showed a reduction in thickness relative to axon diameters. Remarkably, multifocal axonal damage occurred in the corpus callosum. Thereby, debris accumulated between axolemma and inner myelin surface and axons collapsed, although myelin sheaths remained present. These anomalies of myelinated axons were already present in juvenile mice but aggravated in adulthood. Together, loss of CNS peroxisomal metabolism both affects myelin sheaths and axonal integrity possibly via independent pathways.