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Background: Understanding patterns of association between CSF phosphorylated tau (p-tau) species and clinical disease severity will aid Alzheimer’s disease (AD) diagnosis and treatment. Objective: To evaluate changes in tau phosphorylation ratios to brain imaging (amyloid PET, [18F]GTP1 PET, and MRI) and cognition across clinical stages of AD in two different cohorts. Methods: A mass spectrometry (MS)-based method was used to evaluate the relationship between p-tau/tau phosphorylation ratios on 11 sites in CSF and AD pathology measured by tau PET ([18F]GTP1) and amyloid PET ([18F]florbetapir or [18F]florbetaben). Cohort A included cognitively normal amyloid negative (n = 6) and positive (n = 5) individuals, and amyloid positive prodromal (n = 13), mild (n = 12), and moderate AD patients (n = 10); and Cohort B included amyloid positive prodromal (n = 24) and mild (n = 40) AD patients. Results: In this cross-sectional analysis, we identified clusters of phosphosites with different profiles of phosphorylation ratios across stages of disease. Eight of 11 investigated sites were hyperphosphorylated and associated with SUVR measures from [18F]GTP1 and amyloid PET. Novel sites 111, 153, and 208 may be relevant biomarkers for AD diagnosis to complement tau hyperphosphorylation measures on previously established sites 181, 205, 217, and 231. Hypophosphorylation was detected on residues 175, 199, and 202, and was inversely associated with [18F]GTP1 and amyloid PET. Conclusion: Hyperphosphorylated and hypophosphorylated forms of tau are associated with AD pathologies, and due to their different site-specific profiles, they may be used in combination to assist with staging of disease.

Background The role and implementation of tau PET imaging for predicting subsequent cognitive decline in Alzheimer’s disease (AD) remains uncertain. This study was designed to evaluate the relationship between baseline [18F]GTP1 tau PET and subsequent longitudinal change across multiple cognitive measures over 18 months. Methods Our analyses incorporated data from 67 participants, including cognitively normal controls (n = 10) and β-amyloid (Aβ)-positive individuals ([18F] florbetapir Aβ PET) with prodromal (n = 26), mild (n = 16), or moderate (n = 15) AD. Baseline measurements included cortical volume (MRI), tau burden ([18F]GTP1 tau PET), and cognitive assessments [Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR), 13-item version of the Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog13), and Repeatable Battery for the Assessment of Neuropsychological Status (RBANS)]. Cognitive assessments were repeated at 6-month intervals over an 18-month period. Associations between baseline [18F]GTP1 tau PET indices and longitudinal cognitive performance were assessed via univariate (Spearman correlations) and multivariate (linear mixed effects models) approaches. The utility of potential prognostic tau PET cut points was assessed with ROC curves. Results Univariate analyses indicated that greater baseline [18F]GTP1 tau PET signal was associated with faster rates of subsequent decline on the MMSE, CDR, and ADAS-Cog13 across regions of interest (ROIs). In multivariate analyses adjusted for baseline age, cognitive performance, cortical volume, and Aβ PET SUVR, the prognostic performance of [18F]GTP1 SUVR was most robust in the whole cortical gray ROI. When AD participants were dichotomized into low versus high tau subgroups based on baseline [18F]GTP1 PET standardized uptake value ratios (SUVR) in the temporal (cutoff = 1.325) or whole cortical gray (cutoff = 1.245) ROIs, high tau subgroups demonstrated significantly more decline on the MMSE, CDR, and ADAS-Cog13. Conclusions Our results suggest that [18F]GTP1 tau PET represents a prognostic biomarker in AD and are consistent with data from other tau PET tracers. Tau PET imaging may have utility for identifying AD patients at risk for more rapid cognitive decline and for stratification and/or enrichment of participant selection in AD clinical trials. Trial registration ClinicalTrials.gov NCT02640092. Registered on December 28, 2015

Background and purpose Develop a translational assay of Transient Receptor Potential Ankyrin 1 (TRPA1) activity for use as a preclinical and clinical biomarker. Experimental approach Allyl isothiocyanate (AITC), capsaicin or citric acid were applied to ears of wildtype and Trpa1-knock out (Trpa1 KO) rats, and changes in dermal blood flow (DBF) were measured by laser speckle contrast imaging. In humans, the DBF, pain and itch responses to 5-20% AITC applied to the forearm were measured and safety was evaluated. Reproducibility of the DBF, pain and itch responses to topically applied 10% and 15% AITC were assessed at two visits separated by 13-15 days. DBF changes were summarized at 5-minute intervals as areas under the curve (AUC) and maxima. Intraclass correlation coefficient (ICC) was calculated to assess arm-arm and period-period reproducibility. Key results AITC- and citric acid-induced DBF were significantly reduced in Trpa1 KO rats compared to wildtype (90 ± 2% and 65 ± 11% reduction, respectively), whereas capsaicin response did not differ. In humans, each AITC concentration significantly increased DBF compared to vehicle with the maximal increase occurring 5 minutes post application. Ten percent and 15% AITC were selected as safe and effective stimuli. AUC from 0 to 5 minutes was the most reproducible metric of AITC-induced DBF across arms (ICC = 0.92) and periods (ICC = 0.85). Subject-reported pain was more reproducible than itch across visits (ICC = 0.76 vs 0.17, respectively). Conclusion and implications AITC-induced DBF is a suitable target engagement biomarker of TRPA1 activity for preclinical and clinical studies of TRPA1 antagonists.

SUMMARY NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely un-known. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer’s disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12–20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments., Graphical Abstract, In Brief Hanson et al. examine the therapeutic effects of enhancing GluN2A-subunit-containing NMDAR function in Dravet syndrome and Alzheimer’s disease mice. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in both disease models. GluN2A NMDAR enhancers may benefit brain disorders with network hypersynchrony and cognitive impairments.

The regional relationships between tau positron emission tomography (PET) imaging and cognitive impairment in Alzheimer's disease (AD) remain uncertain. We examined cross-sectional associations between cognitive performance, cerebral uptake of the novel tau PET tracer [18F]GTP1, and other neuroimaging indices ([18F]florbetapir amyloid PET, magnetic resonance imaging) in 71 participants with normal cognition, prodromal AD, or AD dementia. Greater [18F]GTP1 uptake was seen with increasing clinical severity and correlated with poorer cognition. [18F]GTP1 uptake and cortical volume (but not [18F]florbetapir uptake) were independently associated with cognitive performance, particularly within the temporal lobe. Delayed memory was more specifically associated with temporal [18F]GTP1 uptake; other domains correlated with a broader range of regional [18F]GTP1 uptake. These data confirm that [18F]GTP1 tau PET uptake significantly correlates with cognitive performance in AD, but regional correlations between performance in non-memory cognitive domains were less specific than reported by tau PET imaging studies that included participants with atypical focal cortical AD syndromes. Tau PET imaging may have utility as a surrogate biomarker for clinical AD progression in therapeutic trials of disease-modifying interventions.

Neurofibrillary tangles (NFTs), consisting of intracellular aggregates of the tau protein, are a pathological hallmark of Alzheimer’s disease (AD). Here we report the identification and initial characterization of Genentech Tau Probe 1 ([18F]GTP1), a small-molecule PET probe for imaging tau pathology in AD patients. Autoradiography using human brain tissues from AD donors and protein binding panels were used to determine [18F]GTP1 binding characteristics. Stability was evaluated in vitro and in vivo in mice and rhesus monkey. In the clinic, whole-body imaging was performed to assess biodistribution and dosimetry. Dynamic [18F]GTP1 brain imaging and input function measurement were performed on two separate days in 5 β-amyloid plaque positive (Aβ+) AD and 5 β-amyloid plaque negative (Aβ-) cognitive normal (CN) participants. Tracer kinetic modeling was applied and reproducibility was evaluated. SUVR was calculated and compared to [18F]GTP1-specific binding parameters derived from the kinetic modeling. [18F]GTP1 performance in a larger cross-sectional group of 60 Aβ+ AD participants and ten (Aβ- or Aβ+) CN was evaluated with images acquired 60 to 90 min post tracer administration. [18F]GTP1 exhibited high affinity and selectivity for tau pathology with no measurable binding to β-amyloid plaques or MAO-B in AD tissues, or binding to other tested proteins at an affinity predicted to impede image data interpretation. In human, [18F]GTP1 exhibited favorable dosimetry and brain kinetics, and no evidence of defluorination. [18F]GTP1-specific binding was observed in cortical regions of the brain predicted to contain tau pathology in AD and exhibited low (< 4%) test-retest variability. SUVR measured in the 60 to 90-min interval post injection correlated with tracer-specific binding (slope = 1.36, r2 = 0.98). Furthermore, in a cross-sectional population, the degree of [18F]GTP1-specific binding increased with AD severity and could differentiate diagnostic cohorts. [18F]GTP1 is a promising PET probe for the study of tau pathology in AD.

Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte–macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases., AhYoung, Eckard et al. show that the expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates the levels of histamine and serotonin in mast cell granules. This study reveals an important physiological function of NSP4 in mast cell-mediated vascular leakage in mice, establishing NSP4 as a potential therapeutic target for mast cell-dependent immune disorders.

Kidney volume is greatly affected in several renal diseases. Precise and automatic segmentation of the kidney can help determine kidney size and evaluate renal function. Fully convolutional neural networks have been used to segment organs from large biomedical 3D images. While these networks demonstrate state-of-the-art segmentation performances, they do not immediately translate to small foreground objects, small sample sizes, and anisotropic resolution in MRI datasets. In this paper we propose a new framework to address some of the challenges for segmenting 3D MRI. These methods were implemented on preclinical MRI for segmenting kidneys in an animal model of lupus nephritis. Our implementation strategy is twofold: 1) to utilize additional MRI diffusion images to detect the general kidney area, and 2) to reduce the 3D U-Net kernels to handle small sample sizes. Using this approach, a Dice similarity coefficient of 0.88 was achieved with a limited dataset of n=196. This segmentation strategy with careful optimization can be applied to various renal injuries or other organ systems.

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

Significance Multiple myeloma (MM) is a lethal malignancy arising from plasma cells. MM cells experience endoplasmic reticulum (ER) stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding protein-folding and secretion capacity, while supporting proteasomal degradation of ER misfolded proteins. IRE1α elaborates these functions by deploying a cytoplasmic kinase–RNase module to activate the transcription factor XBP1s. Although IRE1α has been implicated in MM, its validity as a potential therapeutic target—particularly as a kinase—has been unclear. Using genetic and pharmacologic disruption, we demonstrate that the IRE1α–XBP1s pathway is critical for MM tumor growth. We further show that the kinase domain of IRE1α is an effective and safe potential small-molecule target for MM therapy., Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase–endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α–XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138− cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Microglia are specialized brain macrophages that play numerous roles in tissue homeostasis and response to injury. Colony stimulating factor 1 receptor (CSF1R) is a receptor tyrosine kinase required for the development, maintenance, and proliferation of microglia. Here we show that in adult mice peripheral dosing of function-blocking antibodies to the two known ligands of CSF1R, CSF1, and IL-34, can deplete microglia differentially in white and gray matter regions of the brain, respectively. The regional patterns of depletion correspond to the differential expression of CSF1 and IL-34. In addition, we show that while CSF1 is required to establish microglia in the developing embryo, both CSF1 and IL-34 are required beginning in early postnatal development. These results not only clarify the roles of CSF1 and IL-34 in microglia maintenance, but also suggest that signaling through these two ligands might support distinct sub-populations of microglia, an insight that may impact drug development for neurodegenerative and other diseases.

To date, there is no validated fluid biomarker for tau pathology in Alzheimer’s disease, with contradictory results from studies evaluating the correlation between phosphorylated tau in CSF with tau PET imaging. Tau protein is subjected to proteolytic processing into fragments before being secreted to the CSF. A recent study suggested that tau cleavage after amino acid 368 by asparagine endopeptidase (AEP) is upregulated in Alzheimer’s disease. We used immunoprecipitation followed by mass spectrometric analyses to evaluate the presence of tau368 species in CSF. A novel Simoa® assay for quantification of tau368 in CSF was developed, while total tau (t-tau) was measured by ELISA and the presence of tau368 in tangles was evaluated using immunohistochemistry. The diagnostic utility of tau368 was first evaluated in a pilot study (Alzheimer’s disease = 20, control = 20), then in a second cohort where the IWG-2 biomarker criteria were applied (Alzheimer’s disease = 37, control = 45), and finally in a third cohort where the correlation with 18F-GTP1 tau PET was evaluated (Alzheimer’s disease = 38, control = 11). The tau368/t-tau ratio was significantly decreased in Alzheimer’s disease (P < 0.001) in all cohorts. Immunohistochemical staining demonstrated that tau fragments ending at 368 are present in tangles. There was a strong negative correlation between the CSF tau368/t-tau ratio and 18F-GTP1 retention. Our data suggest that tau368 is a tangle-enriched fragment and that the CSF ratio tau368/t-tau reflects tangle pathology. This novel tau biomarker could be used to improve diagnosis of Alzheimer’s disease and to facilitate the development of drug candidates targeting tau pathology. Furthermore, future longitudinal studies will increase our understanding of tau pathophysiology in Alzheimer’s disease and other tauopathies.

The regional relationships between tau positron emission tomography (PET) imaging and cognitive impairment in Alzheimer's disease (AD) remain uncertain. We examined cross-sectional associations between cognitive performance, cerebral uptake of the novel tau PET tracer [

18F-AV-1451 is currently the most widely used of several experimental tau PET tracers. The objective of this study was to evaluate 18F-AV-1451 binding with full kinetic analysis using a metabolite-corrected arterial input function and to compare parameters derived from kinetic analysis with SUV ratio (SUVR) calculated over different imaging time intervals. Methods:18F-AV-1451 PET brain imaging was completed in 16 subjects: 4 young healthy volunteers (YHV), 4 aged healthy volunteers (AHV), and 8 Alzheimer disease (AD) subjects. Subjects were imaged for 3.5 h, with arterial blood samples obtained throughout. PET data were analyzed using plasma and reference tissue-based methods to estimate the distribution volume, binding potential (BPND), and SUVR. BPND and SUVR were calculated using the cerebellar cortex as a reference region and were compared across the different methods and across the 3 groups (YHV, AHV, and AD). Results: AD demonstrated increased 18F-AV-1451 retention compared with YHV and AHV based on both invasive and noninvasive analyses in cortical regions in which paired helical filament tau accumulation is expected in AD. A correlation of R2 > 0.93 was found between BPND (130 min) and SUVR-1 at all time intervals. Cortical SUVR curves reached a relative plateau around 1.0-1.2 for YHV and AHV by approximately 50 min, but increased in AD by up to approximately 20% at 110-130 min and approximately 30% at 160-180 min relative to 80-100 min. Distribution volume (130 min) was lower by 30%-35% in the YHV than AHV. Conclusion: Our data suggest that although 18F-AV-1451 SUVR curves do not reach a plateau and are still increasing in AD, an SUVR calculated over an imaging window of 80-100 min (as currently used in clinical studies) provides estimates of paired helical filament tau burden in good correlation with BPND, whereas SUVR sensitivity to regional cerebral blood changes needs further investigation.

Age-related macular degeneration (AMD), a leading cause of vision impairment in the ageing population, is characterized by irreversible loss of retinal pigment epithelial (RPE) cells and photoreceptors and can be associated with choroidal neovascularization. Mononuclear phagocytes are often present in AMD lesions, but the processes that direct myeloid cell recruitment remain unclear. Here, we identify IL-33 as a key regulator of inflammation and photoreceptor degeneration after retina stress or injury. IL-33+ Müller cells were more abundant and IL-33 cytokine was elevated in advanced AMD cases compared with age-matched controls with no AMD. In rodents, retina stress resulted in release of bioactive IL-33 that in turn increased inflammatory chemokine and cytokine expression in activated Müller cells. Deletion of ST2, the IL-33 receptor α chain, or treatment with a soluble IL-33 decoy receptor significantly reduced release of inflammatory mediators from Müller cells, inhibited accumulation of mononuclear phagocytes in the outer retina, and protected photoreceptor rods and cones after a retina insult. This study demonstrates a central role for IL-33 in regulating mononuclear phagocyte recruitment to the photoreceptor layer and positions IL-33 signaling as a potential therapeutic target in macular degenerative diseases.

The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aβ reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138− cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1α inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1α with kinase-based small-molecule inhibitors in MM.Significance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1α mitigates ER stress by expanding the ER’s protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1α elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1α as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1α-XBP1s pathway plays a critical role in MM growth. We further show that IRE1α’s kinase domain is an effective and safe potential small-molecule target for MM therapy.

Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

Summary Excitatory synapses occur mainly on dendritic spines, and spine density is usually correlated with the strength of excitatory synaptic transmission. We report that Nr4a1 , an activity-inducible gene encoding a nuclear receptor, regulates the density and distribution of dendritic spines in CA1 pyramidal neurons. Nr4a1 overexpression resulted in elimination of the majority of spines; however, postsynaptic densities were preserved on dendritic shafts, and the strength of excitatory synaptic transmission was unaffected, showing that excitatory synapses can be dissociated from spines. mRNA expression profiling studies suggest that Nr4a1-mediated transcriptional regulation of the actin cytoskeleton contributes to this effect. Under conditions of chronically elevated activity, when Nr4a1 was induced, Nr4a1 knockdown increased the density of spines and PSDs specifically at the distal ends of dendrites. Thus, Nr4a1 is a key component of an activity-induced transcriptional program that regulates the density and distribution of spines and synapses.

In the developing brain, initial neuronal projections are formed through extensive growth and branching of developing axons, but many branches are later pruned to sculpt the mature pattern of connections. Despite its widespread occurrence, the mechanisms controlling pruning remain incompletely characterized. Based on pharmacological and biochemical analysisin vitroand initial genetic analysisin vivo, prior studies implicated a pathway involving binding of the Amyloid Precursor Protein (APP) to Death Receptor 6 (DR6) and activation of a downstream caspase cascade in axonal pruning. Here, we further test their involvement in pruningin vivoand their mechanism of action through extensive genetic and biochemical analysis. Genetic deletion ofDR6was previously shown to impair pruning of retinal axonsin vivo. We show that genetic deletion ofAPPsimilarly impairs pruning of retinal axonsin vivoand provide evidence that APP and DR6 act cell autonomously and in the same pathway to control pruning. Prior analysis had suggested that β-secretase cleavage of APP and binding of an N-terminal fragment of APP to DR6 is required for their actions, but further genetic and biochemical analysis reveals that β-secretase activity is not required and that high-affinity binding to DR6 requires a more C-terminal portion of the APP ectodomain. These results provide direct support for the model that APP and DR6 function cell autonomously and in the same pathway to control pruningin vivoand raise the possibility of alternate mechanisms for how APP and DR6 control pruning.

High-affinity transferrin receptor (TfR) bispecific antibodies facilitate trafficking of TfR to lysosomes and induce TfR degradation to decrease the ability of TfR to mediate BBB transcytosis., Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and β-secretase (BACE1 [β-amyloid cleaving enzyme-1]) traverse the blood–brain barrier (BBB) and effectively reduce brain amyloid β levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.

Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.

Loss of dual leucine zipper kinase results in attenuated JNK/c-Jun stress response pathway activation and reduced neuronal degeneration after kainic acid–induced excitotoxic seizures., Excessive glutamate signaling is thought to underlie neurodegeneration in multiple contexts, yet the pro-degenerative signaling pathways downstream of glutamate receptor activation are not well defined. We show that dual leucine zipper kinase (DLK) is essential for excitotoxicity-induced degeneration of neurons in vivo. In mature neurons, DLK is present in the synapse and interacts with multiple known postsynaptic density proteins including the scaffolding protein PSD-95. To examine DLK function in the adult, DLK-inducible knockout mice were generated through Tamoxifen-induced activation of Cre-ERT in mice containing a floxed DLK allele, which circumvents the neonatal lethality associated with germline deletion. DLK-inducible knockouts displayed a modest increase in basal synaptic transmission but had an attenuation of the JNK/c-Jun stress response pathway activation and significantly reduced neuronal degeneration after kainic acid–induced seizures. Together, these data demonstrate that DLK is a critical upstream regulator of JNK-mediated neurodegeneration downstream of glutamate receptor hyper-activation and represents an attractive target for the treatment of indications where excitotoxicity is a primary driver of neuronal loss.

Axon degeneration initiated by trophic factor withdrawal shares many features with programmed cell death, but many prior studies discounted a role for caspases in this process, particularly Caspase-3. Recently, Caspase-6 was implicated based on pharmacological and knockdown evidence, and we report here that genetic deletion ofCaspase-6indeed provides partial protection from degeneration. However, we find at a biochemical level that Caspase-6 is activated effectively only by Caspase-3 but not other “upstream” caspases, prompting us to revisit the role of Caspase-3.In vitro, we show that genetic deletion ofCaspase-3is fully protective against sensory axon degeneration initiated by trophic factor withdrawal, but not injury-induced Wallerian degeneration, and we define a biochemical cascade from prosurvival Bcl2 family regulators to Caspase-9, then Caspase-3, and then Caspase-6. Only low levels of active Caspase-3 appear to be required, helping explain why its critical role has been obscured in prior studies.In vivo,Caspase-3andCaspase-6-knockout mice show a delay in developmental pruning of retinocollicular axons, thereby implicating both Caspase-3 and Caspase-6 in axon degeneration that occurs as a part of normal development.

In addition to being a hallmark of neurodegenerative disease, axon degeneration is used during development of the nervous system to prune unwanted connections. In development, axon degeneration is tightly regulated both temporally and spatially. Here, we provide evidence that degeneration cues are transduced through various kinase pathways functioning in spatially distinct compartments to regulate axon degeneration. Intriguingly, glycogen synthase kinase-3 (GSK3) acts centrally, likely modulating gene expression in the cell body to regulate distally restricted axon degeneration. Through a combination of genetic and pharmacological manipulations, including the generation of an analog-sensitive kinase allele mutant mouse for GSK3β, we show that the β isoform of GSK3, not the α isoform, is essential for developmental axon pruningin vitroandin vivo. Additionally, we identify thedleu2/mir15a/16-1cluster, previously characterized as a regulator of B-cell proliferation, and the transcription factortbx6, as likely downstream effectors of GSK3β in axon degeneration.

SummaryThe proapoptotic death receptor DR5 has been studied extensively in cancer cells, but its action in the tumor microenvironment is not well defined. Here, we uncover a role for DR5 signaling in tumor endothelial cells (ECs). We detected DR5 expression in ECs within tumors but not normal tissues. Treatment of tumor-bearing mice with an oligomeric form of the DR5 ligand Apo2L/TRAIL induced apoptosis in tumor ECs, collapsing blood vessels and reducing tumor growth: Vascular disruption and antitumor activity required DR5 expression on tumor ECs but not malignant cells. These results establish a therapeutic paradigm for proapoptotic receptor agonists as selective tumor vascular disruption agents, providing an alternative, perhaps complementary, strategy to their use as activators of apoptosis in malignant cells.

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V1 sector that catalyzes ATP hydrolysis and the transmembrane Vo sector responsible for proton translocation. The transmembrane Vo complex directs the complex to different membranes, but also has been proposed to have roles independent of the V1 sector. However, the roles of the V1 sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V1 B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V1 sector, in addition to the Vo sector, is required in exocytic and endocytic pathways.

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface. In Caenorhabditis elegans, secretory membranous organelles (MO) fuse with the plasma membrane to transform spermatids into fertilization-competent spermatozoa. We show that, like the acrosomal vesicle of mammalian sperm, MOs undergo acidification during development. Treatment of spermatids with the V-ATPase inhibitor bafilomycin blocks both MO acidification and formation of functional spermatozoa. There are several spermatogenesis-defective mutants that cause defects in MO morphogenesis, including spe-5. We determined that spe-5, which is on chromosome I, encodes one of two V-ATPase B paralogous subunits. The spe-5 null mutant is viable but sterile because it forms arrested, multi-nucleate spermatocytes. Immunofluorescence with a SPE-5-specific monoclonal antibody shows that SPE-5 expression begins in spermatocytes and is found in all subsequent stages of spermatogenesis. Most SPE-5 is discarded into the residual body during spermatid budding, but a small amount remains in budded spermatids where it localizes to MOs as a discrete dot. The other V-ATPase B subunit is encoded by vha-12, which is located on the X chromosome. Usually, spe-5 mutants are self-sterile in a wild-type vha-12 background. However, an extrachromosomal transgene containing wild-type vha-12 driven by its own promoter allows spe-5 mutant hermaphrodites to produce progeny, indicating that VHA-12 can at least partially substitute for SPE-5. Others have shown that the X chromosome is transcriptionally silent in the male germline, so expression of the autosomally located spe-5 gene ensures that a V-ATPase B subunit is present during spermatogenesis.

Summary Signaling events that regulate central nervous system (CNS) angiogenesis and blood-brain barrier (BBB) formation are only beginning to be elucidated. By evaluating the gene expression profile of mouse vasculature, we identified DR6/TNFRSF21 and TROY/TNFRSF19 as regulators of CNS-specific angiogenesis in both zebrafish and mice. Furthermore, these two death receptors interact both genetically and physically and are required for vascular endothelial growth factor (VEGF)-mediated JNK activation and subsequent human brain endothelial sprouting in vitro. Increasing beta-catenin levels in brain endothelium upregulate DR6 and TROY, indicating that these death receptors are downstream target genes of Wnt/beta-catenin signaling, which has been shown to be required for BBB development. These findings define a role for death receptors DR6 and TROY in CNS-specific vascular development.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1–42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

Objective— To quantitatively compare aortic curvature and motion with resulting aneurysm location, direction of expansion, and pathophysiological features in experimental abdominal aortic aneurysms (AAAs). Methods and Results— MRI was performed at 4.7 T with the following parameters: (1) 3D acquisition for vessel geometry and (2) 2D cardiac-gated acquisition to quantify luminal motion. Male 24-week-old mice were imaged before and after AAA formation induced by angiotensin II (AngII)–filled osmotic pump implantation or infusion of elastase. AngII-induced AAAs formed near the location of maximum abdominal aortic curvature, and the leftward direction of expansion was correlated with the direction of suprarenal aortic motion. Elastase-induced AAAs formed in a region of low vessel curvature and had no repeatable direction of expansion. AngII significantly increased mean blood pressure (22.7 mm Hg, P P Conclusion— The direction of AngII aneurysm expansion correlated with the direction of motion, medial elastin dissection, and adventitial remodeling. Anterior infrarenal aortic motion correlated with medial elastin degradation in elastase-induced aneurysms. Results from both models suggest a relationship between aneurysm pathological features and aortic geometry and motion.

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Clathrin is a coat protein involved in vesicle budding from several membrane-bound compartments within the cell. Here we present an analysis of a temperature-sensitive (ts) mutant of clathrin heavy chain (CHC) in a multicellular animal. As expected Caenorhabditis elegans chc-1 ( b1025ts) mutant animals are defective in receptor-mediated endocytosis and arrest development soon after being shifted to the restrictive temperature. Steady-state clathrin levels in these mutants are reduced by more than 95% at all temperatures. Hub interactions and membrane associations are lost at the restrictive temperature. chc-1 ( b1025ts ) animals become paralyzed within minutes of exposure to the restrictive temperature because of a defect in the nervous system. Surprisingly synaptic vesicle number is not reduced in chc-1(b1025ts ) animals. Consistent with the normal number of vesicles, postsynaptic miniature currents occur at normal frequencies. Taken together, these results indicate that a high level of CHC activity is required for receptor-mediated endocytosis in nonneuronal cells but is largely dispensable for maintenance of synaptic vesicle pools.

Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families: physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In Caenorhabditis elegans , only a single member of each of these families exists. These three nematode TVPs colocalize to the same vesicular compartment when expressed in mammalian cells, suggesting that they could serve overlapping functions. To examine their function, C. elegans null mutants were isolated for each gene, and a triple mutant was generated. Surprisingly, these animals develop normally and exhibit normal neuronal architecture and synaptic contacts. In addition, functions of the motor and sensory systems are normal as determined by pharmacological, chemotaxis, and thermotaxis assays. Finally, direct electrophysiological analysis of the neuromuscular junction revealed no phenotype in the TVP mutants. We therefore conclude that TVPs are not needed for the basic neuronal machinery and instead may contribute to subtle higher order functions.

Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing unc-18 mutants of the nematode Caenorhabditis elegans. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked vesicles at the active zone in unc-18 mutants, suggesting that UNC-18 functions, directly or indirectly, as a facilitator of vesicle docking.

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

Extensive evidence implicates GluN2B-containing NMDA receptors (GluN2B-NMDARs) in excitotoxic-insult-induced neurodegeneration and amyloid β (Aβ)-induced synaptic dysfunction. Therefore, inhibiting GluN2B-NMDARs would appear to be a potential therapeutic strategy to provide neuroprotection and improve cognitive function in Alzheimer's disease (AD). However, there are no reports of long-term in vivo treatment of AD mouse models with GluN2B antagonists. We used piperidine18 (Pip18), a potent and selective GluN2B-NMDAR antagonist with favorable pharmacokinetic properties, for long-term dosing in AD mouse models. Reduced freezing behavior in Tg2576 mice during fear conditioning was partially reversed after subchronic (17 d) Pip18 treatment. However, analysis of freezing behavior in different contexts indicated that this increased freezing likely involves elevated anxiety or excessive memory generalization in both nontransgenic (NTG) and Tg2576 mice. In PS2APP mice chronically fed with medicated food containing Pip18 for 4 months, spatial learning and memory deficits were not rescued, plaque-associated spine loss was not affected, and synaptic function was not altered. At the same time, altered open field activity consistent with increased anxiety and degraded performance in an active avoidance task were observed in NTG after chronic treatment. These results indicate that long-term treatment with a GluN2B-NMDAR antagonist does not provide a disease-modifying benefit and could cause cognitive liabilities rather than symptomatic benefit in AD mouse models. Therefore, these results challenge the expectation of the therapeutic potential for GluN2B-NMDAR antagonists in AD.