Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons.

The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation.

Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal re-pigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.

Computational modeling of multiscale collateral blood supply in a whole-brain-scale arterial network.

The cerebral arterial network covering the brain cortex has multiscale anastomosis structures with sparse intermediate anastomoses (O[102] µm in diameter) and dense pial networks (O[101] µm in diameter). Recent studies indicate that collateral blood supply by cerebral arterial anastomoses has an essential role in the prognosis of acute ischemic stroke caused by large vessel occlusion. However, the physiological importance of these multiscale morphological properties-and especially of intermediate anastomoses-is poorly understood because of innate structural complexities. In this study, a computational model of multiscale anastomoses in whole-brain-scale cerebral arterial networks was developed and used to evaluate collateral blood supply by anastomoses during middle cerebral artery occlusion. Morphologically validated cerebral arterial networks were constructed by combining medical imaging data and mathematical modeling. Sparse intermediate anastomoses were assigned between adjacent main arterial branches; the pial arterial network was modeled as a dense network structure. Blood flow distributions in the arterial network during middle cerebral artery occlusion simulations were computed. Collateral blood supply by intermediate anastomoses increased sharply with increasing numbers of anastomoses and provided one-order-higher flow recoveries to the occluded region (15%-30%) compared with simulations using a pial network only, even with a small number of intermediate anastomoses (≤10). These findings demonstrate the importance of sparse intermediate anastomoses, which are generally considered redundant structures in cerebral infarction, and provide insights into the physiological significance of the multiscale properties of arterial anastomoses.

Bidirectional Regulation of Motor Circuits Using Magnetogenetic Gene Therapy.

Regulating the activity of discrete neuronal populations in living mammals after delivery of modified ion channels can be used to map functional circuits and potentially treat neurological diseases. Here we report a novel suite of magnetogenetic tools, based on a single anti-ferritin nanobody-TRPV1 receptor fusion protein, which regulated neuronal activity in motor circuits when exposed to magnetic fields. AAV-mediated delivery of a cre-dependent nanobody-TRPV1 calcium channel into the striatum of adenosine 2a (A2a) receptor-cre driver mice led to restricted expression within D2 neurons, resulting in motor freezing when placed in a 3T MRI or adjacent to a transcranial magnetic stimulation (TMS) device. Functional imaging and fiber photometry both confirmed focal activation of the target region in response to the magnetic fields. Expression of the same construct in the striatum of wild-type mice along with a second injection of an AAVretro expressing cre into the globus pallidus led to similar circuit specificity and motor responses. Finally, a mutation was generated to gate chloride and inhibit neuronal activity. Expression of this variant in subthalamic nucleus (STN) projection neurons in PitX2-cre parkinsonian mice resulted in reduced local c-fos expression and a corresponding improvement in motor rotational behavior during magnetic field exposure. These data demonstrate that AAV delivery of magnetogenetic constructs can bidirectionally regulate activity of specific neuronal circuits non-invasively in vivo using clinically available devices for both preclinical analysis of circuit effects on behavior and potential human clinical translation.

Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation.

Vitiligo is an autoimmune skin disease caused by cutaneous melanocyte loss. Although phototherapy and T cell suppression therapy have been widely used to induce epidermal repigmentation, full pigmentation recovery is rarely achieved due to our poor understanding of the cellular and molecular mechanisms governing this process. Here, we identify unique melanocyte stem cell (McSC) epidermal migration rates between male and female mice, which is due to sexually dimorphic cutaneous inflammatory responses generated by ultra-violet B exposure. Using genetically engineered mouse models, and unbiased bulk and single-cell mRNA sequencing approaches, we determine that manipulating the inflammatory response through cyclooxygenase and its downstream prostaglandin product regulates McSC proliferation and epidermal migration in response to UVB exposure. Furthermore, we demonstrate that a combinational therapy that manipulates both macrophages and T cells (or innate and adaptive immunity) significantly promotes epidermal melanocyte re-population. With these findings, we propose a novel therapeutic strategy for repigmentation in patients with depigmentation conditions such as vitiligo.

Vascular oxidative stress causes neutrophil arrest in brain capillaries, leading to decreased cerebral blood flow and contributing to memory impairment in a mouse model of Alzheimer’s disease.

INTRODUCTION: In this study, we explore the role of oxidative stress produced by NOX2-containing NADPH oxidase as a molecular mechanism causing capillary stalling and cerebral blood flow deficits in the APP/PS1 mouse model of AD. METHODS: We inhibited NOX2 in APP/PS1 mice by administering a 10 mg/kg dose of the peptide inhibitor gp91-ds-tat i.p., for two weeks. We used in vivo two-photon imaging to measure capillary stalling, penetrating arteriole flow, and vascular inflammation. We also characterized short-term memory function and gene expression changes in cerebral microvessels. RESULTS: We found that after NOX2 inhibition capillary stalling, as well as parenchymal and vascular inflammation, were significantly reduced. In addition, we found a significant increase in penetrating arteriole flow, followed by an improvement in short-term memory, and downregulation of inflammatory gene expression pathways. DISCUSSION: Oxidative stress is a major mechanism leading to microvascular dysfunction in AD, and represents an important therapeutic target.

Other iatrogenic immunodeficiency-associated lymphoproliferative disorders in a patient with anti-melanoma differentiation-associated gene 5-positive dermatomyositis: A case report and systematic literature review.

A 58-year-old man with anti-melanoma differentiation-associated gene 5-positive dermatomyositis (MDA5-DM) developed Epstein-Barr virus (EBV)-associated malignant lymphoma as other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPD) during the combined immunosuppressive therapy of high-dose prednisolone, tacrolimus, and intravenous cyclophosphamide for MDA5-DM. Serum EBV DNA was detected, and EBV-encoded small RNA was positive in the tissue sample of LPD, indicating that EBV reactivation contributed to the pathogenesis of LPD in our case. The patient underwent chemotherapy, including rituximab, promptly after discontinuation of tacrolimus and cyclophosphamide, resulting in complete remission of the malignant lymphoma, and MDA5-DM has not recurred with 3.5 mg/d of prednisolone monotherapy. We reviewed 19 cases of OIIA-LPD in patients with idiopathic inflammatory myopathies and herein report the first case of MDA5-DM complicated with OIIA-LPD. Among the 19 patients, 7 showed regression of LPD only following withdrawal of immunosuppressants, 9 took chemotherapy for LPD, and 5 died. It should be noted that patients with MDA5-DM-associated rapidly progressive interstitial lung disease could develop OIIA-LPD because they receive aggressive immunosuppressive therapy.

Airway epithelial STAT3 inhibits allergic inflammation via upregulation of stearoyl-CoA desaturase 1.

BACKGROUND: Airway epithelial cells (AECs) play a crucial role in the induction and development of allergic inflammation through the development and activation of immune cells, including Th2 cells and ILC2s. Recent studies have revealed that STAT3 expressed in epithelial cells protects against pathogens and maintains homeostasis in the intestine. However, the roles of STAT3 in airway epithelium are poorly understood. Therefore, we sought to elucidate the roles of airway epithelial STAT3 in allergic airway inflammation. METHODS: Allergic airway inflammation was induced by intratracheal administration of house dust mite (HDM) extract in doxycycline-induced AEC-specific STAT3-deficient (STAT3-cKO) mice and their genetic control (STAT3-WT) mice. Airway inflammation was evaluated by flow cytometric analysis of bronchoalveolar lavage fluid cells and histological analysis of the lung. Purified airway epithelial cells were analyzed by quantitative PCR and RNA-sequencing (RNA-seq). RESULTS: HDM-induced airway inflammation was exacerbated in STAT3-cKO mice compared with STAT3-WT mice. RNA-seq analyses revealed that Scd1, coding stearoyl-CoA desaturase 1, was most significantly upregulated in HDM-treated STAT3-WT mice compared to HDM-treated STAT3-cKO mice. Notably, the administration of an SCD1 inhibitor exacerbated HDM-induced airway inflammation. AECs of HDM-treated STAT3-cKO mice and those of HDM-treated SCD1 inhibitor-injected mice shared 45 differentially expressed genes (DEGs). Gene enrichment analysis of the DEGs revealed that the enriched ontology clusters included fatty acid biosynthetic process and regulation of lipid biosynthetic process, suggesting the involvement of the STAT3-SCD1-lipid metabolism axis in suppressing allergic inflammation. CONCLUSIONS: STAT3 is crucial for suppressing HDM-induced allergic airway inflammation, possibly inducing SCD1 expression in AECs.

Neurological and Inflammatory Effects of Radio Frequency and Cryoablation in a Rat Sciatic Nerve Model of Submucosal Nerve Ablation.

BACKGROUND: Minimally-invasive ablation with radio frequency (RF) and cryoablation have been widely adopted to treat conditions with aberrant neural activity such as excessive mucus production in rhinitis, but neurological and inflammatory effects on treated tissues are poorly understood. OBJECTIVE: To gain an understanding of the physiological changes caused by nerve ablation using RF and cryoablation devices. METHODS: Using clinical devices for rhinitis treatment that ablate nerves with access from the nasal cavity, we applied temperature-controlled RF and cryoablation to rat sciatic nerves. To model the ablation through mucosal tissue similarly to the rhinitis procedure, RF ablation and cryoablation were applied through a layer of muscle. RESULTS: Both ablation techniques induced acute and sustained neurodegeneration visualized with histological sections at two days and one month after treatment. After both treatments, rats showed a change in muscle tone, but small increases in sensitivity measured by a von Frey test were only observed 2 days after cryoablation and one month after the RF ablation. Both treatments caused reductions in nerve conduction velocity at one month after treatment. Inflammation in treated nerves and surrounding tissues that persisted to one month. CONCLUSIONS: The two neurolytic devices used in the clinic work similarly by axonal disintegration and which leads to disruption of electrical signals. The data suggest that these methods are effective methods of nerve ablation that could be used to treat diseases related to elevated neuron activity such as rhinitis.

Navigating neurophotonics, words of wisdom: an interview with Professor David Kleinfeld.

David Kleinfeld, the Dr. George Feher Endowed Chair in Experimental Biophysics at UC San Diego, shares his views on training and best practices in neurophotonics, offering words of wisdom for building and strengthening our scientific community.

Differential regulation of progranulin derived granulin peptides.

BACKGROUND: Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and is processed into individual granulin peptides in the lysosome. However, very little is known about the levels and regulations of individual granulin peptides due to the lack of specific antibodies. RESULTS: Here we report the generation and characterization of antibodies specific to each granulin peptide. We found that the levels of granulins C, E and F are regulated differently  compared to granulins A and B in various tissues. The levels of PGRN and granulin peptides vary in different brain regions and the ratio between granulins and PGRN is highest in the cortical region in the adult male mouse brain. Granulin-A is localized in the lysosome in both neurons and microglia and its levels in microglia increase under pathological conditions. Interestingly,  the levels of granulin A in microglia change correspondingly with PGRN in response to stroke but not demyelination. Furthermore, deficiency of lysosomal proteases and the PGRN binding partner prosaposin leads to alterations in the ratios between individual granulin peptides. Granulins B, C and E are heavily glycosylated and the glycosylation patterns can be regulated. CONCLUSION: Our results support that the levels of individual granulin peptides are differentially regulated under physiological and pathological conditions and provide novel insights into how granulin peptides function in the lysosome.

VEGF signalling causes stalls in brain capillaries and reduces cerebral blood flow in Alzheimer's mice.

Increased incidence of stalled capillary blood flow caused by adhesion of leucocytes to the brain microvascular endothelium leads to a 17% reduction of cerebral blood flow and exacerbates short-term memory loss in multiple mouse models of Alzheimer's disease. Here, we report that vascular endothelial growth factor (VEGF) signalling at the luminal side of the brain microvasculature plays an integral role in the capillary stalling phenomenon of the APP/PS1 mouse model. Administration of the anti-mouse VEGF-A164 antibody, an isoform that inhibits blood-brain barrier hyperpermeability, reduced the number of stalled capillaries within an hour of injection, leading to an immediate increase in average capillary blood flow but not capillary diameter. VEGF-A inhibition also reduced the overall endothelial nitric oxide synthase protein concentrations, increased occludin levels and decreased the penetration of circulating Evans Blue dye across the blood-brain barrier into the brain parenchyma, suggesting increased blood-brain barrier integrity. Capillaries prone to neutrophil adhesion after anti-VEGF-A treatment also had lower occludin concentrations than flowing capillaries. Taken together, our findings demonstrate that VEGF-A signalling in APP/PS1 mice contributes to aberrant endothelial nitric oxide synthase /occludin-associated blood-brain barrier permeability, increases the incidence of capillary stalls, and leads to reductions in cerebral blood flow. Reducing leucocyte adhesion by inhibiting luminal VEGF signalling may provide a novel and well-tolerated strategy for improving brain microvascular blood flow in Alzheimer's disease patients.

Genetically engineered mice for combinatorial cardiovascular optobiology.

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Associations of ultrasound-based inflammation patterns with peripheral innate lymphoid cell populations, serum cytokines/chemokines, and treatment response to methotrexate in rheumatoid arthritis and spondyloarthritis.

OBJECTIVES: We aimed to explore the associations of musculoskeletal inflammation patterns with peripheral blood innate lymphoid cell (ILC) populations, serum cytokines/chemokines, and treatment response to methotrexate in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA). METHODS: We enrolled 100 patients with either RA or SpA and performed ultrasound to evaluate power Doppler signals for synovitis (52 joint regions), tenosynovitis (20 tendons), and enthesitis (44 sites). We performed clustering analysis using unsupervised random forest based on the multi-axis ultrasound information and classified the patients into groups. We identified and counted ILC1-3 populations in the peripheral blood by flow cytometry and also measured the serum levels of 20 cytokines/chemokines. We also determined ACR20 response at 3 months in 38 patients who began treatment with methotrexate after study assessment. RESULTS: Synovitis was more prevalent and severe in RA than in SpA, whereas tenosynovitis and enthesitis were comparable between RA and SpA. Patients were classified into two groups which represented synovitis-dominant and synovitis-nondominant inflammation patterns. While peripheral ILC counts were not significantly different between RA and SpA, they were significantly higher in the synovitis-nondominant group than in the synovitis-dominant group (ILC1-3: p = 0.0007, p = 0.0061, and p = 0.0002, respectively). On the other hand, clustering of patients based on serum cytokines/chemokines did not clearly correspond either to clinical diagnoses or to synovitis-dominant/nondominant patterns. The synovitis-dominant pattern was the most significant factor that predicted clinical response to methotrexate (p = 0.0065). CONCLUSIONS: Musculoskeletal inflammation patterns determined by ultrasound are associated with peripheral ILC counts and could predict treatment response to methotrexate.

Causes and consequences of baseline cerebral blood flow reductions in Alzheimer's disease.

Reductions of baseline cerebral blood flow (CBF) of ∼10-20% are a common symptom of Alzheimer's disease (AD) that appear early in disease progression and correlate with the severity of cognitive impairment. These CBF deficits are replicated in mouse models of AD and recent work shows that increasing baseline CBF can rapidly improve the performance of AD mice on short term memory tasks. Despite the potential role these data suggest for CBF reductions in causing cognitive symptoms and contributing to brain pathology in AD, there remains a poor understanding of the molecular and cellular mechanisms causing them. This review compiles data on CBF reductions and on the correlation of AD-related CBF deficits with disease comorbidities (e.g. cardiovascular and genetic risk factors) and outcomes (e.g. cognitive performance and brain pathology) from studies in both patients and mouse models, and discusses several potential mechanisms proposed to contribute to CBF reductions, based primarily on work in AD mouse models. Future research aimed at improving our understanding of the importance of and interplay between different mechanisms for CBF reduction, as well as at determining the role these mechanisms play in AD patients could guide the development of future therapies that target CBF reductions in AD.

High fat diet worsens Alzheimer's disease-related behavioral abnormalities and neuropathology in APP/PS1 mice, but not by synergistically decreasing cerebral blood flow.

Obesity is linked to increased risk for and severity of Alzheimer's disease (AD). Cerebral blood flow (CBF) reductions are an early feature of AD and are also linked to obesity. We recently showed that non-flowing capillaries, caused by adhered neutrophils, contribute to CBF reduction in mouse models of AD. Because obesity could exacerbate the vascular inflammation likely underlying this neutrophil adhesion, we tested links between obesity and AD by feeding APP/PS1 mice a high fat diet (Hfd) and evaluating behavioral, physiological, and pathological changes. We found trends toward poorer memory performance in APP/PS1 mice fed a Hfd, impaired social interactions with either APP/PS1 genotype or a Hfd, and synergistic impairment of sensory-motor function in APP/PS1 mice fed a Hfd. The Hfd led to increases in amyloid-beta monomers and plaques in APP/PS1 mice, as well as increased brain inflammation. These results agree with previous reports showing obesity exacerbates AD-related pathology and symptoms in mice. We used a crowd-sourced, citizen science approach to analyze imaging data to determine the impact of the APP/PS1 genotype and a Hfd on capillary stalling and CBF. Surprisingly, we did not see an increase in the number of non-flowing capillaries or a worsening of the CBF deficit in APP/PS1 mice fed a Hfd as compared to controls, suggesting that capillary stalling is not a mechanistic link between a Hfd and increased severity of AD in mice. Reducing capillary stalling by blocking neutrophil adhesion improved CBF and short-term memory function in APP/PS1 mice, even when fed a Hfd.

Impact of Chirality on Hydrogen-Bonded Supramolecular Assemblies and Photoconductivity of Diketopyrrolopyrrole Derivatives.

Hydrogen bonds can efficiently guide the self-assembly of organic materials, enabling to tune the properties of the aggregation processes. In the case of π-conjugated materials, several parameters such as temperature, concentration and solvent can be used to modify the aggregation state while tuning the optoelectronic properties. Chirality can be included within the impacting parameters due to the differences in molecular packing. Here, chiral and achiral thiophene-capped diketopyrrolopyrrole derivatives were designed and synthesized containing amide bonds, with the aim to study the interplay between chiral assemblies and their stabilization through hydrogen-bonding. Differences in aggregation properties were observed with spectroscopy and microscopy, and a contactless microwave-based technique was used to study their intrinsic charge carrier mobility. The positive role of hydrogen-bonding has been highlighted and the differences between chiral and achiral compounds have been elucidated.

Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

Microvessel occlusions alter amyloid-beta plaque morphology in a mouse model of Alzheimer's disease.

Vascular dysfunction is correlated to the incidence and severity of Alzheimer's disease. In a mouse model of Alzheimer's disease (APP/PS1) using in vivo, time-lapse, multiphoton microscopy, we found that occlusions of the microvasculature alter amyloid-beta (Aß) plaques. We used several models of vascular injury that varied in severity. Femtosecond laser-induced occlusions in single capillaries generated a transient increase in small, cell-sized, Aß deposits visualized with methoxy-X04, a label of fibrillar Aß. After occlusions of penetrating arterioles, some plaques changed morphology, while others disappeared, and some new plaques appeared within a week after the lesion. Antibody labeling of Aß revealed a transient increase in non-fibrillar Aß one day after the occlusion that coincided with the disappearance of methoxy-X04-labeled plaques. Four days after the lesion, anti-Aß labeling decreased and only remained in patches unlabeled by methoxy-X04 near microglia. Histology in two additional models, sparse embolic occlusions from intracarotid injections of beads and infarction from photothrombosis, demonstrated increased labeling intensity in plaques after injury. These results suggest that microvascular lesions can alter the deposition and clearance of Aß and confirm that Aß plaques are dynamic structures, complicating the interpretation of plaque burden as a marker of Alzheimer's disease progression.