DNA Hypermethylation and Unstable Repeat Diseases: A Paradigm of Transcriptional Silencing to Decipher the Basis of Pathogenic Mechanisms.

Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.

Protocol for the Characterization of the Cytosine-Adenine-Guanine Tract and Flanking Polymorphisms in Machado-Joseph Disease: Impact on Diagnosis and Development of Gene-Based Therapies.

Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs.

High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation.

Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.

Targeting Ubiquitin Proteasome Pathway with Traditional Chinese Medicine for Treatment of Spinocerebellar Ataxia Type 3.

Nine autosomal dominant spinocerebellar ataxias (SCAs) are caused by an abnormal expansion of CAG trinucleotide repeats that encodes a polyglutamine (polyQ) tract within different genes. Accumulation of aggregated mutant proteins is a common feature of polyQ diseases, leading to progressive neuronal dysfunction and degeneration. SCA type 3 (SCA3), the most common form of SCA worldwide, is characterized by a CAG triplet expansion in chromosome 14q32.1 ATXN3 gene. As accumulation of the mutated polyQ protein is a possible initial event in the pathogenic cascade, clearance of aggregated protein by ubiquitin proteasome system (UPS) has been proposed to inhibit downstream detrimental events and suppress neuronal cell death. In this study, Chinese herbal medicine (CHM) extracts were studied for their proteasome-activating, polyQ aggregation-inhibitory and neuroprotective effects in GFPu and ATXN3/Q 75 -GFP 293/SH-SY5Y cells. Among the 14 tested extracts, 8 displayed increased proteasome activity, which was confirmed by 20S proteasome activity assay and analysis of ubiquitinated and fused GFP proteins in GFPu cells. All the eight extracts displayed good aggregation-inhibitory potential when tested in ATXN3/Q 75 -GFP 293 cells. Among them, neuroprotective effects of five selected extracts were shown by analyses of polyQ aggregation, neurite outgrowth, caspase 3 and proteasome activities, and ATXN3-GFP, ubiquitin, BCL2 and BAX protein levels in neuronal differentiated ATXN3/Q 75 -GFP SH-SY5Y cells. Finally, enhanced proteasome function, anti-oxidative activity and neuroprotection of catalpol, puerarin and daidzein (active constituents of Rehmannia glutinosa and Pueraria lobata) were demonstrated in GFPu and/or ATXN3/Q 75 -GFP 293/SH-SY5Y cells. This study may have therapeutic implication in polyQ-mediated disorders.

Dissecting Pathogenetic Mechanisms and Therapeutic Strategies in Drosophila Models of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1), the most common cause of adult-onset muscular dystrophy, is autosomal dominant, multisystemic disease with characteristic symptoms including myotonia, heart defects, cataracts and testicular atrophy. DM1 disease is being successfully modelled in Drosophila allowing to identify and validate new pathogenic mechanisms and potential therapeutic strategies. Here we provide an overview of insights gained from fruit fly DM1 models, either: (i) fundamental with particular focus on newly identified gene deregulations and their link with DM1 symptoms; or (ii) applied via genetic modifiers and drug screens to identify promising therapeutic targets.

Brain-Targeting Delivery of Two Peptidylic Inhibitors for Their Combination Therapy in Transgenic Polyglutamine Disease Mice via Intranasal Administration.

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 µmol/kg) and L1P3V8 (6 µmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.

High-dose 1,25-dihydroxyvitamin D supplementation elongates the lifespan of Huntington's disease transgenic mice.

Huntington's disease is an autosomal dominant progressive neurodegenerative disease, which results in a decreased quality of life and an early death. A high prevalence of vitamin D deficiency was first described in a 2013 study in patients with manifest Huntington's disease, where serum vitamin D level was found to be associated with motor capabilities of the patients. Our objective was to investigate the effect of a high-dose vitamin D3 supplementation on a transgenic mouse model of Huntington's disease. Our study was performed on N171-82Q Huntington's disease transgenic mice in age- and gender-matched groups. We collected data on the motor state and survival of the mice. The results demonstrate that though vitamin D3 had no effect on the motor performance of transgenic mice, but significantly increased the lifespan of transgenic animals (Kaplan-Meier survival curves: vehicle-supplemented group: 73 (67-94) days vs. vitamin D3-supplemented group: 101 (74-109) days, p=0.048 Mantel-Cox log rank test). Further investigations are needed to determine whether a neuroprotective or a general corroborative effect of vitamin D leads to the measured effect. Our findings support the potential influence of vitamin D deficiency on the disease course and propose that vitamin D may be an effective supplementary treatment to beneficially influence clinical features of Huntington's disease.

Altered selenium status in Huntington's disease: neuroprotection by selenite in the N171-82Q mouse model.

Disruption of redox homeostasis is a prominent feature in the pathogenesis of Huntington's disease (HD). Selenium an essential element nutrient that modulates redox pathways and has been reported to provide protection against both acute neurotoxicity (e.g. methamphetamine) and chronic neurodegeneration (e.g. tauopathy) in mice. The objective of our study was to investigate the effect of sodium selenite, an inorganic form of selenium, on behavioral, brain degeneration and biochemical outcomes in the N171-82Q Huntington's disease mouse model. HD mice, which were supplemented with sodium selenite from 6 to 14 weeks of age, demonstrated increased motor endurance, decreased loss of brain weight, decreased mutant huntingtin aggregate burden and decreased brain oxidized glutathione levels. Biochemical studies revealed that selenite treatment reverted HD-associated changes in liver selenium and plasma glutathione in N171-82Q mice and had effects on brain selenoprotein transcript expression. Further, we found decreased brain selenium content in human autopsy brain. Taken together, we demonstrate a decreased selenium phenotype in human and mouse HD and additionally show some protective effects of selenite in N171-82Q HD mice. Modification of selenium metabolism results in beneficial effects in mouse HD and thus may represent a therapeutic strategy.

Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expanded CAG repeats in the huntingtin (HTT) gene. Although several palliative treatments are available, there is currently no cure and patients generally die 10-15 y after diagnosis. Several promising approaches for HD therapy are currently in development, including RNAi and antisense analogs. We developed a complementary strategy to test repression of mutant HTT with zinc finger proteins (ZFPs) in an HD model. We tested a "molecular tape measure" approach, using long artificial ZFP chains, designed to bind longer CAG repeats more strongly than shorter repeats. After optimization, stable ZFP expression in a model HD cell line reduced chromosomal expression of the mutant gene at both the protein and mRNA levels (95% and 78% reduction, respectively). This was achieved chromosomally in the context of endogenous mouse HTT genes, with variable CAG-repeat lengths. Shorter wild-type alleles, other genomic CAG-repeat genes, and neighboring genes were unaffected. In vivo, striatal adeno-associated virus viral delivery in R6/2 mice was efficient and revealed dose-dependent repression of mutant HTT in the brain (up to 60%). Furthermore, zinc finger repression was tested at several levels, resulting in protein aggregate reduction, reduced decline in rotarod performance, and alleviation of clasping in R6/2 mice, establishing a proof-of-principle for synthetic transcription factor repressors in the brain.

Behavioral and in vivo electrophysiological evidence for presymptomatic alteration of prefrontostriatal processing in the transgenic rat model for huntington disease.

Cognitive decline precedes motor symptoms in Huntington disease (HD). A transgenic rat model for HD carrying only 51 CAG repeats recapitulates the late-onset HD phenotype. Here, we assessed prefrontostriatal function in this model through both behavioral and electrophysiological assays. Behavioral examination consisted in a temporal bisection task within a supra-second range (2 vs.8 s), which is thought to involve prefrontostriatal networks. In two independent experiments, the behavioral analysis revealed poorer temporal sensitivity as early as 4 months of age, well before detection of overt motor deficits. At a later symptomatic age, animals were impaired in their temporal discriminative behavior. In vivo recording of field potentials in the dorsomedial striatum evoked by stimulation of the prelimbic cortex were studied in 4- to 5-month-old rats. Input/output curves, paired-pulse function, and plasticity induced by theta-burst stimulation (TBS) were assessed. Results showed an altered plasticity, with higher paired-pulse facilitation, enhanced short-term depression, as well as stronger long-term potentiation after TBS in homozygous transgenic rats. Results from the heterozygous animals mostly fell between wild-type and homozygous transgenic rats. Our results suggest that normal plasticity in prefrontostriatal circuits may be necessary for reliable and precise timing behavior. Furthermore, the present study provides the first behavioral and electrophysiological evidence of a presymptomatic alteration of prefrontostriatal processing in an animal model for Huntington disease and suggests that supra-second timing may be the earliest cognitive dysfunction in HD.

Expanded-polyglutamine huntingtin protein suppresses the secretion and production of a chemokine (CCL5/RANTES) by astrocytes.

Huntington's disease (HD) is a hereditary neurological disease caused by expended CAG repeats in the HD gene, which codes for a protein called Huntingtin (Htt). The resultant mutant Huntingtin (mHtt) forms aggregates in neurons and causes neuronal dysfunction. In astrocytes, the largest population of brain cells, mHtt also exists. We report herein that astrocyte-conditioned medium (ACM) collected from astrocytes of R6/2 mice (a mouse model of HD) caused primary cortical neurons to grow less-mature neurites, migrate more slowly, and exhibit lower calcium influx after depolarization than those maintained in wild-type (WT) ACM. Using a cytokine antibody array and ELISA assays, we demonstrated that the amount of a chemokine [chemokine (C-C motif) ligand 5 (CCL5)/regulated on activation normal T cell expressed and secreted (RANTES)] released by R6/2 astrocytes was much less than that by WT astrocytes. When cortical neurons were treated with the indicated ACM, supplementation with recombinant CCL5/RANTES ameliorated the neuronal deficiency caused by HD-ACM, whereas removing CCL5/RANTES from WT-ACM using an anti-CCL5/RANTES antibody mimicked the effects evoked by HD-ACM. Quantitative PCR and promoter analyses demonstrated that mHtt hindered the activation of the CCL5/RANTES promoter by reducing the availability of nuclear factor kappaB-p65 and, hence, reduced the transcript level of CCL5/RANTES. Moreover, ELISA assays and immunocytochemical staining revealed that mHtt retained the residual CCL5/RANTES inside R6/2 astrocytes. In line with the above findings, elevated cytosolic CCL5/RANTES levels were also observed in the brains of two mouse models of HD [R6/2 and Hdh((CAG)150)] and human HD patients. These findings suggest that mHtt hinders one major trophic function of astrocytes which might contribute to the neuronal dysfunction of HD.

DM1 CTG expansions affect insulin receptor isoforms expression in various tissues of transgenic mice.

Myotonic dystrophy (DM1) is a dominant autosomal multisystemic disorder caused by the expansion of an unstable CTG trinucleotide repeat in the 3' untranslated region of the DMPK gene. Nuclear accumulation of the enlarged CUG-containing DMPK transcripts has a deleterious effect on the regulation of alternative splicing of some RNAs and has a central role in causing the symptoms of DM1. In particular, Insulin Receptor (IR) mRNA splicing defects have been observed in the muscle of DM1 patients. In this study, we have investigated IR splicing in insulin-responsive tissues (i.e. skeletal muscles, adipose tissue, liver) and pancreas and we have studied glucose metabolism in mice carrying the human genomic DM1 region with expanded (>350 CTG) or normal (20 CTG) repeats and in wild-type mice. Mice carrying DM1 expansions displayed a tissue- and age-dependent abnormal regulation of IR mRNA splicing in all the tissues that we investigated. Furthermore, these mice showed a basal hyperglycemia and glucose intolerance which disappeared with age. Our findings show that deregulation of IR splicing due to the DM1 mutation can occur in different mouse tissues, suggesting that CTG repeat expansions might also result in IR misplicing not only in muscles but also in other tissues in DM1 patients.

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis.

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.

Rational selection of small molecules that increase transcription through the GAA repeats found in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA.

Genetic and pharmacological suppression of polyglutamine-dependent neuronal dysfunction in Caenorhabditis elegans.

The identification of disease genes for several neurodegenerative illnesses has allowed for the development of disease models in experimental organisms. We discuss our approach to studying Huntington's disease, the best characterized of the polyglutamine (polyQ) expansion disorders. We have developed a system in Caenorhabditis elegans to study the effects of (polyQ)-dependent neuronal dysfunction at the resolution of two neurons in screening for genetic and pharmacological suppression. Our data suggest that C. elegans might be instructive in searching for targets and active compounds against polyglutamine neuronal toxicity.

Topography of cerebral atrophy in early Huntington's disease: a voxel based morphometric MRI study.

OBJECTIVES: To analyse grey matter changes in early stages of Huntington's disease using magnetic resonance imaging (MRI) and the technique of voxel based morphometry (VBM). METHODS: Forty four patients with a molecularly confirmed clinical diagnosis of Huntington's disease based on the presence of motor signs were included in the study. Patients were clinically rated using the Unified Huntington's Disease Rating Scale; all were in early clinical stages of the disease (that is, Shoulson stages I and II). High resolution volume rendering MRI scans (MP-RAGE) were acquired. MRI data were volumetrically analysed in comparison to an age matched normal database by VBM, using statistical parametric mapping (SPM99). RESULTS: In Huntington's disease, robust regional decreases in grey matter density (p<0.001, corrected for multiple comparisons)-that is, atrophy-were found bilaterally in striatal areas as well as in the hypothalamus and the opercular cortex, and unilaterally in the right paracentral lobule. The topography of striatal changes corresponded to the dorso-ventral gradient of neuronal loss described in neuropathological studies. Stratification according to clinical severity showed a more widespread involvement extending into the ventral aspects of the striatum in the group of more severely affected patients. CONCLUSIONS: The topography of cerebral volume changes associated with Huntington's disease can be mapped using VBM. It can be shown that cerebral grey matter changes co-vary with clinical severity and CAG repeat length.

A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.

Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.

A family with spinocerebellar ataxia type 8 expansion and vitamin E deficiency ataxia.

BACKGROUND: Ataxia with vitamin E deficiency is a recessive autosomal neurodegenerative disorder resembling the Friedreich ataxia phenotype but is due to mutations in the alpha-tocopherol transfer protein (TTPA) gene. In a recent article, we described a patient with ataxia carrying reduced serum vitamin E levels and showing CTA/CTG expansions of 320 triplet repeats in the SCA8 gene. OBJECTIVES: To perform a screening of the TTPA gene in the patient and to evaluate the effects of treatment with vitamin E on the patient's neurologic disturbances. PATIENT AND METHODS: We performed a single-strand conformation polymorphism and nucleotide sequence analysis of the 5 exons of the TTPA gene in the patient's family members. RESULTS: The results indicated the patient to be a compound heterozygote for 2 mutations (in exon 3), each transmitted by one of the 2 parents, yielding a nonfunctional protein. CONCLUSIONS: We describe for the first time, to our knowledge, a mutated form of the TTPA gene in a patient also carrying an expansion in the SCA8 gene. The lack of improvement in the patient's symptoms on supplementation with alpha-tocopherol suggests that the SCA8 mutations may act in the neurodegeneration process, worsening the neurologic signs caused by the vitamin E deficit, and it could be speculated that the co-occurrence of mutant alleles for 2 distinct loci may influence the clinical course of the disease.

Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles.

Huntington's disease (HD) is a late onset neurodegenerative disorder caused by a CAG/polyglutamine (polyQ) repeat expansion. PolyQ aggregates can be detected in the nuclei and processes of neurons in HD patients and mouse models prior to the onset of symptoms. The misfolding and aggregation pathway is an important therapeutic target. To better test the efficacy of aggregation inhibitors, we have developed an organotypic slice culture system. We show here that the formation of polyQ aggregates in hippocampal slices established from the R6/2 mouse follows the same prescribed sequence as occurs in vivo. Using this assay, we show that Congo red and chrysamine G can modulate aggregate formation, but show complex dose-response curves. Oral administration of creatine has been shown to delay the onset of all aspects of the phenotype and neuropathology in R6/2 mice. We show here that creatine can similarly inhibit aggregate formation in the slice culture assay.