Large-scale gene expression changes in APP/PSEN1 and GFAP mutation models exhibit high congruence with Alzheimer's disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with both genetic and non-genetic causes. Animal research models are available for a multitude of diseases and conditions affecting the central nervous system (CNS), and large-scale CNS gene expression data exist for many of these. Although there are several models specifically for AD, each recapitulates different aspects of the human disease. In this study we evaluate over 500 animal models to identify those with CNS gene expression patterns matching human AD datasets. Approaches included a hypergeometric based scoring system that rewards congruent gene expression patterns but penalizes discordant gene expression patterns. The top two models identified were APP/PS1 transgenic mice expressing mutant APP and PSEN1, and mice carrying a GFAP mutation that is causative of Alexander disease, a primary disorder of astrocytes in the CNS. The APP/PS1 and GFAP models both matched over 500 genes moving in the same direction as in human AD, and both had elevated GFAP expression and were highly congruent with one another. Also scoring highly were the 5XFAD model (with five mutations in APP and PSEN1) and mice carrying CK-p25, APP, and MAPT mutations. Animals with the APOE3 and 4 mutations combined with traumatic brain injury ranked highly. Bulbectomized rats scored high, suggesting anosmia could be causative of AD-like gene expression. Other matching models included the SOD1G93A strain and knockouts for SNORD116 (Prader-Willi mutation), GRID2, INSM1, XBP1, and CSTB. Many top models demonstrated increased expression of GFAP, and results were similar across multiple human AD datasets. Heatmap and Uniform Manifold Approximation Plot results were consistent with hypergeometric ranking. Finally, some gene manipulation models, including for TYROBP and ATG7, were identified with reversed AD patterns, suggesting possible neuroprotective effects. This study provides insight for the pathobiology of AD and the potential utility of available animal models.

Quantitative diffusion imaging and genotype-by-sex interactions in a rat model of Alexander disease.

PURPOSE: The clinical diagnosis and classification of Alexander disease (AxD) relies in part on qualitative neuroimaging biomarkers; however, these biomarkers fail to distinguish and discriminate different subtypes of AxD, especially in the presence of overlap in clinical symptoms. To address this gap in knowledge, we applied neurite orientation dispersion and density imaging (NODDI) to an innovative CRISPR-Cas9 rat genetic model of AxD to gain quantitative insights into the neural substrates and brain microstructural changes seen in AxD and to potentially identify novel quantitative NODDI biomarkers of AxD. METHODS: Multi-shell DWI of age- and sex-matched AxD and wild-type Sprague Dawley rats (n = 6 per sex per genotype) was performed and DTI and NODDI measures calculated. A 3 × 2 × 2 analysis of variance model was used to determine the effect of genotype, biological sex, and laterality on quantitative measures of DTI and NODDI across regions of interest implicated in AxD. RESULTS: There is a significant effect of genotype in the amygdala, hippocampus, neocortex, and thalamus in measures of both DTI and NODDI brain microstructure. A genotype by biological sex interaction was identified in DTI and NODDI measures in the corpus callosum, hippocampus, and neocortex. CONCLUSION: We present the first application of NODDI to the study of AxD using a rat genetic model of AxD. Our analysis identifies alterations in NODDI and DTI measures to large white matter tracts and subcortical gray nuclei. We further identified genotype by sex interactions, suggesting a possible role for biological sex in the neuropathogenesis of AxD.

STAT3 Drives GFAP Accumulation and Astrocyte Pathology in a Mouse Model of Alexander Disease.

Alexander disease (AxD) is caused by mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament expressed by astrocytes in the central nervous system. AxD-associated mutations cause GFAP aggregation and astrogliosis, and GFAP is elevated with the astrocyte stress response, exacerbating mutant protein toxicity. Studies in mouse models suggest disease severity is tied to Gfap expression levels, and signal transducer and activator of transcription (STAT)-3 regulates Gfap during astrocyte development and in response to injury and is activated in astrocytes in rodent models of AxD. In this report, we show that STAT3 is also activated in the human disease. To determine whether STAT3 contributes to GFAP elevation, we used a combination of genetic approaches to knockout or reduce STAT3 activation in AxD mouse models. Conditional knockout of Stat3 in cells expressing Gfap reduced Gfap transactivation and prevented protein accumulation. Astrocyte-specific Stat3 knockout in adult mice with existing pathology reversed GFAP accumulation and aggregation. Preventing STAT3 activation reduced markers of reactive astrocytes, stress-related transcripts, and microglial activation, regardless of disease stage or genetic knockout approach. These results suggest that pharmacological inhibition of STAT3 could potentially reduce GFAP toxicity and provide a therapeutic benefit in patients with AxD.

Anastasis Drives Senescence and Non-Cell Autonomous Neurodegeneration in the Astrogliopathy Alexander Disease.

Anastasis is a recently described process in which cells recover after late-stage apoptosis activation. The functional consequences of anastasis for cells and tissues are not clearly understood. Using Drosophila, rat and human cells and tissues, including analyses of both males and females, we present evidence that glia undergoing anastasis in the primary astrogliopathy Alexander disease subsequently express hallmarks of senescence. These senescent glia promote non-cell autonomous death of neurons by secreting interleukin family cytokines. Our findings demonstrate that anastasis can be dysfunctional in neurologic disease by inducing a toxic senescent population of astroglia.SIGNIFICANCE STATEMENT Under some conditions cells otherwise destined to die can be rescued just before death in a process called anastasis, or "rising from the dead." The fate and function of cells undergoing a near death experience is not well understood. Here, we find that in models and patient cells from Alexander disease, an important brain disorder in which glial cells promote neuronal dysfunction and death, anastasis of astrocytic glia leads to secretion of toxic signaling molecules and neurodegeneration. These studies demonstrate a previously unexpected deleterious consequence of rescuing cells on the brink of death and suggest therapeutic strategies for Alexander disease and related disorders of glia.

Alexander disease: models, mechanisms, and medicine.

Alexander disease is a primary disorder of astrocytes caused by gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), which lead to protein aggregation and a reactive astrocyte response, with devastating effects on the central nervous system. Over the past two decades since the discovery of GFAP as the culprit, several cellular and animal models have been generated, and much has been learned about underlying mechanisms contributing to the disease. Despite these efforts, many aspects of Alexander disease have remained enigmatic, particularly the initiating events in GFAP accumulation and astrocyte pathology, the relation between astrocyte dysfunction and myelin deficits, and the variability in age of onset and disease severity. More recent work in both old and new models has begun to address these complex questions and identify new therapeutics that finally offer the promise of effective treatment.

Antisense therapy in a rat model of Alexander disease reverses GFAP pathology, white matter deficits, and motor impairment.

Alexander disease (AxD) is a devastating leukodystrophy caused by gain-of-function mutations in GFAP, and the only available treatments are supportive. Recent advances in antisense oligonucleotide (ASO) therapy have demonstrated that transcript targeting can be a successful strategy for human neurodegenerative diseases amenable to this approach. We have previously used mouse models of AxD to show that Gfap-targeted ASO suppresses protein accumulation and reverses pathology; however, the mice have a mild phenotype with no apparent leukodystrophy or overt clinical features and are therefore limited for assessing functional outcomes. In this report, we introduce a rat model of AxD that exhibits hallmark pathology with GFAP aggregation in the form of Rosenthal fibers, widespread astrogliosis, and white matter deficits. These animals develop normally during the first postnatal weeks but fail to thrive after weaning and develop severe motor deficits as they mature, with about 14% dying of unknown cause between 6 and 12 weeks of age. In this model, a single treatment with Gfap-targeted ASO provides long-lasting suppression, reverses GFAP pathology, and, depending on age of treatment, prevents or mitigates white matter deficits and motor impairment. In this report, we characterize an improved animal model of AxD with myelin pathology and motor impairment, recapitulating prominent features of the human disease, and use this model to show that ASO therapy has the potential to not only prevent but also reverse many aspects of disease.

Pexidartinib treatment in Alexander disease model mice reduces macrophage numbers and increases glial fibrillary acidic protein levels, yet has minimal impact on other disease phenotypes.

BACKGROUND: Alexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models. METHODS: To examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed. RESULTS: In AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration. CONCLUSIONS: Together, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.

Type II Alexander disease caused by splicing errors and aberrant overexpression of an uncharacterized GFAP isoform.

Alexander disease results from gain-of-function mutations in the gene encoding glial fibrillary acidic protein (GFAP). At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for ∼90% of GFAP protein. We describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-messenger RNA (mRNA) and result in the upregulation of a previously uncharacterized GFAP lambda isoform (NM_001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM_001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM_001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in the upregulation of the lambda isoform. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing to inform future pathophysiologic and therapeutic study for Alexander disease.

Tissue and cellular rigidity and mechanosensitive signaling activation in Alexander disease.

Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander disease, and possibly also in other brain disorders characterized by gliosis.

Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander disease.

OBJECTIVE: Alexander disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander disease. METHODS: In this study, we use GFAP mutant mouse models of Alexander disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP. RESULTS: Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis. INTERPRETATION: These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for Alexander disease. Ann Neurol 2018;83:27-39.

An In Vivo Pharmacological Screen Identifies Cholinergic Signaling as a Therapeutic Target in Glial-Based Nervous System Disease.

The role that glia play in neurological disease is poorly understood but increasingly acknowledged to be critical in a diverse group of disorders. Here we use a simple genetic model of Alexander disease, a progressive and severe human degenerative nervous system disease caused by a primary astroglial abnormality, to perform an in vivo screen of 1987 compounds, including many FDA-approved drugs and natural products. We identify four compounds capable of dose-dependent inhibition of nervous system toxicity. Focusing on one of these hits, glycopyrrolate, we confirm the role for muscarinic cholinergic signaling in pathogenesis using additional pharmacologic reagents and genetic approaches. We further demonstrate that muscarinic cholinergic signaling works through downstream Gαq to control oxidative stress and death of neurons and glia. Importantly, we document increased muscarinic cholinergic receptor expression in Alexander disease model mice and in postmortem brain tissue from Alexander disease patients, and that blocking muscarinic receptors in Alexander disease model mice reduces oxidative stress, emphasizing the translational significance of our findings. We have therefore identified glial muscarinic signaling as a potential therapeutic target in Alexander disease, and possibly in other gliopathic disorders as well. SIGNIFICANCE STATEMENT: Despite the urgent need for better treatments for neurological diseases, drug development for these devastating disorders has been challenging. The effectiveness of traditional large-scale in vitro screens may be limited by the lack of the appropriate molecular, cellular, and structural environment. Using a simple Drosophila model of Alexander disease, we performed a moderate throughput chemical screen of FDA-approved drugs and natural compounds, and found that reducing muscarinic cholinergic signaling ameliorated clinical symptoms and oxidative stress in Alexander disease model flies and mice. Our work demonstrates that small animal models are valuable screening tools for therapeutic compound identification in complex human diseases and that existing drugs can be a valuable resource for drug discovery given their known pharmacological and safety profiles.

Nitric oxide mediates glial-induced neurodegeneration in Alexander disease.

Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction.

Deficits in adult neurogenesis, contextual fear conditioning, and spatial learning in a Gfap mutant mouse model of Alexander disease.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease.

Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation.

IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²5 in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²5 produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.

GFAP expression as an indicator of disease severity in mouse models of Alexander disease.

AxD (Alexander disease) is a rare disorder caused by heterozygous mutations in GFAP (glial fibrillary acidic protein) resulting in accumulation of the GFAP protein and elevation of Gfap mRNA. To test whether GFAP itself can serve as a biomarker of disease status or progression, we investigated two independent measures of GFAP expression in AxD mouse models, one using a genetic reporter of promoter activity and the other quantifying GFAP protein directly in a manner that could also be employed in human studies. Using a transgenic reporter line that expresses firefly luciferase under the control of the murine Gfap promoter (Gfap-luc), we found that luciferase activity reflected the regional CNS (central nervous system) variability of Gfap mRNA in Gfap(+/+) mice, and increased in mice containing a point mutation in Gfap that mimics a common human mutation in AxD (R239H in the human sequence, and R236H in the murine sequence). In a second set of studies, we quantified GFAP protein in CSF (cerebrospinal fluid) taken from three different AxD mouse models and littermate controls. GFAP levels in CSF were increased in all three AxD models, in a manner corresponding to the concentrations of GFAP in brain. These studies demonstrate that transactivation of the Gfap promoter is an early and sustained indicator of the disease process in the mouse. Furthermore, GFAP in CSF serves as a potential biomarker that is comparable between mouse models and human patients.

The effect of glial fibrillary acidic protein expression on neurite outgrowth from retinal explants in a permissive environment.

BACKGROUND: Increased expression of glial fibrillary acidic protein (GFAP) within macroglia is commonly seen as a hallmark of glial activation after damage within the central nervous system, including the retina. The increased expression of GFAP in glia is also considered part of the pathologically inhibitory environment for regeneration of axons from damaged neurons. Recent studies have raised the possibility that reactive gliosis and increased GFAP cannot automatically be assumed to be negative events for the surrounding neurons and that the context of the reactive gliosis is critical to whether neurons benefit or suffer. We utilized transgenic mice expressing a range of Gfap to titrate the amount of GFAP in retinal explants to investigate the relationship between GFAP concentration and the regenerative potential of retinal ganglion cells. FINDINGS: Explants from Gfap-/- and Gfap+/- mice did not have increased neurite outgrowth compared with Gfap+/+ or Gfap over-expressing mice as would be expected if GFAP was detrimental to axon regeneration. In fact, Gfap over-expressing explants had the most neurite outgrowth when treated with a neurite stimulatory media. Transmission electron microscopy revealed that neurites formed bundles, which were surrounded by larger cellular processes that were GFAP positive indicating a close association between growing axons and glial cells in this regeneration paradigm. CONCLUSIONS: We postulate that glial cells with increased Gfap expression support the elongation of new neurites from retinal ganglion cells possibly by providing a scaffold for outgrowth.

Beneficial effects of Nrf2 overexpression in a mouse model of Alexander disease.

Alexander disease is a fatal neurodegenerative disease caused by dominant mutations in glial fibrillary acidic protein (GFAP). The disease is characterized by protein inclusions called Rosenthal fibers within astrocyte cell bodies and processes, and an antioxidant response mediated by the transcription factor Nrf2. We sought to test whether further elevation of Nrf2 would be beneficial in a mouse model of Alexander disease. Forcing overexpression of Nrf2 in astrocytes of R236H GFAP mutant mice decreased GFAP protein in all brain regions examined (olfactory bulb, hippocampus, cerebral cortex, brainstem, cerebellum, and spinal cord) and decreased Rosenthal fibers in olfactory bulb, hippocampus, corpus callosum, and brainstem. Nrf2 overexpression also restored body weights of R236H mice to near wild-type levels. Nrf2 regulates several genes involved in homeostasis of the antioxidant molecule glutathione, and the neuroprotective effects of Nrf2 in other neurological disorders may reflect restoration of glutathione to normal levels. However, glutathione levels in R236H mice were not decreased. Nrf2 overexpression did not change glutathione levels or ratio of reduced to oxidized glutathione (indicative of oxidative stress) in olfactory bulb, where Nrf2 dramatically reduced GFAP. Depletion of glutathione through knock-out of the GCLM (glutamate-cysteine ligase modifier subunit) also did not affect GFAP levels or body weight of R236H mice. These data suggest that the beneficial effects of Nrf2 are not mediated through glutathione.

Genetic ablation of Nrf2/antioxidant response pathway in Alexander disease mice reduces hippocampal gliosis but does not impact survival.

In Alexander disease (AxD) the presence of mutant glial fibrillary acidic protein (GFAP), the major intermediate filament of astrocytes, triggers protein aggregation, with marked induction of a stress response mediated by the transcription factor, Nrf2. To clarify the role of Nrf2 in AxD, we have crossed Gfap mutant and transgenic mouse models into an Nrf2 null background. Deletion of Nrf2 eliminates the phase II stress response normally present in mouse models of AxD, but causes no change in body weight or lifespan, even in a severe lethal model. AxD astrocytes without Nrf2 retain features of reactivity, such as expression of the endothelin-B receptor, but have lower Gfap levels, a decrease in p62 protein and reduced iron accumulation, particularly in hippocampus. Microglial activation, indicated by Iba1 expression, is also diminished. Although the Nrf2 response is generally considered beneficial, these results show that in the context of AxD, loss of the antioxidant pathway has no obvious negative effects, while actually decreasing Gfap accumulation and pathology. Given the attention Nrf2 is receiving as a potential therapeutic target in AxD and other neurodegenerative diseases, it will be interesting to see whether induction of Nrf2, beyond the endogenous response, is beneficial or not in these same models.

Strategies for treatment in Alexander disease.

Alexander disease is a rare and generally fatal disorder of the CNS, originally classified among the leukodystrophies because of the prominent myelin deficits found in young patients. The most common form of this disease affects infants, who often have profound mental retardation and a variety of developmental delays, but later onset forms also occur, sometimes with little or no white matter pathology at all. The pathological hallmark of Alexander disease is the inclusion body, known as Rosenthal fiber, within the cell bodies and processes of astrocytes. Recent genetic studies identified heterozygous missense mutations in glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astrocytes, as the cause of nearly all cases of Alexander disease. These studies have transformed our view of this disorder and opened new directions for investigation and clinical practice, particularly with respect to diagnosis. Mechanisms by which expression of mutant forms of glial fibrillary acidic protein (GFAP) lead to the pleiotropic manifestations of disease (afflicting cell types beyond the ones expressing the mutant gene) are slowly coming into focus. Ideas are beginning to emerge that suggest several compelling therapeutic targets for interventions that might slow or arrest the evolution of the disease. This review will outline the rationale for pursuing these strategies, and highlight some of the critical issues that must be addressed in the planning of future clinical trials.