Transgenic Brain Research Articles

Unspecific binding of cRNA probe to plaques in two mouse models for Alzheimer's disease.

BackgroundAlzheimer’s disease (AD) is characterized by the pathological deposition of amyloid-β (Aβ) protein-containing plaques. Microglia and astrocytes are commonly attracted to the plaques by an unknown mechanism that may involve cell adhesion. One cell adhesion family of proteins, the cadherins, are widely expressed in the central nervous system. Therefore, our study was designed to map the expression of cadherins in AD mouse brains. A particular focus was on plaques because diverse mRNA-species were found in plaques and their surrounding area in brains of AD patients.MethodsIn this study, we used in situ hybridization to visualize cadherin expression in brains of two mouse models for AD (APP/PS1 and APP23).ResultsA variable number of plaques was detected in transgenic brain sections, depending on the probe used. Our first impression was that the cadherin probes visualized specific mRNA expression in plaques and that endogenous staining was unaffected. However, control experiments revealed unspecific binding with sense probes. Further experiments with variations in probe length, probe sequence, molecular tag and experimental procedure lead us to conclude that cRNA probes bind generally and in an unspecific manner to plaques.ConclusionsWe demonstrate unspecific binding of cRNA probes to plaques in two mouse models for AD. The widespread and general staining of the plaques prevented us from studying endogenous expression of cadherins in transgenic brain by in situ hybridization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-016-0065-9) contains supplementary material, which is available to authorized users.

The transcription factor XBP1s restores hippocampal synaptic plasticity and memory by control of the Kalirin-7 pathway in Alzheimer model

Neuronal network dysfunction and cognitive decline constitute the most prominent features of Alzheimer’s disease (AD), although mechanisms causing such impairments are yet to be determined. Here we report that virus-mediated delivery of the active spliced transcription factor X-Box binding protein 1s (XBP1s) in the hippocampus rescued spine density, synaptic plasticity and memory function in a mouse model of AD. XBP1s transcriptionally activated Kalirin-7 (Kal7), a protein that controls synaptic plasticity. In addition, we found reduced levels of Kal7 in primary neurons exposed to Aβ oligomers, transgenic mouse models and human AD brains. Short hairpin RNA-mediated knockdown of Kal7 altered synaptic plasticity and memory formation in naive mice. Further, reduction of endogenous Kal7 compromised the beneficial effects of XBP1s in Alzheimer’s model. Hence, our findings reveal that XBP1s is neuroprotective through a mechanism that engages Kal7 pathway with therapeutic implications in AD pathology.

Brain enhancer activities at the gene-poor 5p14.1 autism-associated locus.

Due to the vast clinical and genetic heterogeneity, identification of causal genetic determinants for autism spectrum disorder (ASD) has proven to be complex. Whereas several dozen ‘rare’ genetic variants for ASD susceptibility have been identified, studies are still underpowered to analyse ‘common’ variants for their subtle effects. A recent application of genome-wide association studies (GWAS) to ASD indicated significant associations with the single nucleotide polymorphisms (SNPs) on chromosome 5p14.1, located in a non-coding region between cadherin10 (CDH10) and cadherin9 (CDH9). Here we apply an in vivo bacterial artificial chromosome (BAC) based enhancer-trapping strategy in mice to scan the gene desert for spatiotemporal cis-regulatory activities. Our results show that the ASD-associated interval harbors the cortical area, striatum, and cerebellum specific enhancers for a long non-coding RNA, moesin pseudogene1 antisense (MSNP1AS) during the brain developing stages. Mouse moesin protein levels are not affected by exogenously expressed human antisense RNAs in our transgenic brains, demonstrating the difficulty in modeling rather smaller effects of common variants. Our first in vivo evidence for the spatiotemporal transcription of MSNP1AS however provides a further support to connect this intergenic variant with the ASD susceptibility.

Oligomers of Amyloid β Prevent Physiological Activation of the Cellular Prion Protein-Metabotropic Glutamate Receptor 5 Complex by Glutamate in Alzheimer Disease

The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling.

Micro-computed tomography in murine models of cerebral cavernous malformations as a paradigm for brain disease

BackgroundCerebral cavernous malformations (CCMs) are hemorrhagic brain lesions, where murine models allow major mechanistic discoveries, ushering genetic manipulations and preclinical assessment of therapies. Histology for lesion counting and morphometry is essential yet tedious and time consuming. We herein describe the application and validations of X-ray micro-computed tomography (micro-CT), a non-destructive technique allowing three-dimensional CCM lesion count and volumetric measurements, in transgenic murine brains. New methodWe hereby describe a new contrast soaking technique not previously applied to murine models of CCM disease. Volumetric segmentation and image processing paradigm allowed for histologic correlations and quantitative validations not previously reported with the micro-CT technique in brain vascular disease. ResultsTwenty-two hyper-dense areas on micro-CT images, identified as CCM lesions, were matched by histology. The inter-rater reliability analysis showed strong consistency in the CCM lesion identification and staging (K=0.89, p<0.0001) between the two techniques. Micro-CT revealed a 29% greater CCM lesion detection efficiency, and 80% improved time efficiency. Comparison with existing methodSerial integrated lesional area by histology showed a strong positive correlation with micro-CT estimated volume (r2=0.84, p<0.0001). ConclusionsMicro-CT allows high throughput assessment of lesion count and volume in pre-clinical murine models of CCM. This approach complements histology with improved accuracy and efficiency, and can be applied for lesion burden assessment in other brain diseases.

The insect central complex as model for heterochronic brain development-background, concepts, and tools.

The adult insect brain is composed of neuropils present in most taxa. However, the relative size, shape, and developmental timing differ between species. This diversity of adult insect brain morphology has been extensively described while the genetic mechanisms of brain development are studied predominantly in Drosophila melanogaster. However, it has remained enigmatic what cellular and genetic mechanisms underlie the evolution of neuropil diversity or heterochronic development. In this perspective paper, we propose a novel approach to study these questions. We suggest using genome editing to mark homologous neural cells in the fly D. melanogaster, the beetle Tribolium castaneum, and the Mediterranean field cricket Gryllus bimaculatus to investigate developmental differences leading to brain diversification. One interesting aspect is the heterochrony observed in central complex development. Ancestrally, the central complex is formed during embryogenesis (as in Gryllus) but in Drosophila, it arises during late larval and metamorphic stages. In Tribolium, it forms partially during embryogenesis. Finally, we present tools for brain research in Tribolium including 3D reconstruction and immunohistochemistry data of first instar brains and the generation of transgenic brain imaging lines. Further, we characterize reporter lines labeling the mushroom bodies and reflecting the expression of the neuroblast marker gene Tc-asense, respectively.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-016-0542-7) contains supplementary material, which is available to authorized users.

Review: Spreading the word: precise animal models and validated methods are vital when evaluating prion-like behaviour of alpha-synuclein.

Synucleinopathies are characterized by abnormal proteinaceous aggregates, mainly composed of fibrillar α-synuclein (α-syn). It is now believed that α-syn can form small aggregates in a restricted number of cells, that propagate to neighbouring cells and seed aggregation of endogenous α-syn, in a 'prion-like manner'. This process could underlie the stereotypical progression of Lewy bodies described by Braak and colleagues across different stages of Parkinson's disease (PD). This prion-like behaviour of α-syn has been recently investigated in animal models of PD or multiple system atrophy (MSA). These models investigate the cell-to-cell transfer of α-syn seeds, or the induction and spreading of α-syn pathology in transgenic or wild-type rodent brain. In this review, we first outline the involvement of α-syn in Lewy body diseases and MSA, and discuss how 'prion-like' mechanisms can contribute to disease. Thereon, we debate the relevance of animal models used to study prion-like propagation. Finally, we review current main histological methods used to assess α-syn pathology both in animal models and in human samples and their relevance to the disease. Specifically, we discuss using α-syn phosphorylated at serine 129 as a marker of pathology, and the novel methods available that allow for more sensitive detection of early pathology, which has relevance for modelling synucleinopathies.

Synphilin-1 attenuates mutant LRRK2-induced neurodegeneration in Parkinson's disease models.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant Parkinsonism with pleomorphic pathology including deposits of aggregated protein and neuronal degeneration. The pathogenesis of LRRK2-linked Parkinson's disease (PD) is not fully understood. Here, using co-immunoprecipitation, we found that LRRK2 interacted with synphilin-1 (SP1), a cytoplasmic protein that interacts with α-synuclein and has implications in PD pathogenesis. LRRK2 interacted with the N-terminus of SP1 whereas SP1 predominantly interacted with the C-terminus of LRRK2, including kinase domain. Co-expression of SP1 with LRRK2 increased LRRK2-induced cytoplasmic aggregation in cultured cells. Moreover, SP1 also attenuated mutant LRRK2-induced toxicity and reduced LRRK2 kinase activity in cultured cells. Knockdown of SP1 by siRNA enhanced LRRK2 neuronal toxicity. In vivo Drosophila studies, co-expression of SP1 and mutant G2019S-LRRK2 in double transgenic Drosophila increased survival and improved locomotor activity. Expression of SP1 protects against G2019S-LRRK2-induced dopamine neuron loss and reduced LRRK2 phosphorylation in double transgenic fly brains. Our findings demonstrate that SP1 attenuates mutant LRRK2-induced PD-like phenotypes and plays a neural protective role.

Aβ Extraction from Murine Brain Homogenates.

This protocol details beta-amyloid (Aβ) extraction from transgenic murine brain homogenates. Specifically, mechanical homogenization of brain tissue and sequential extraction of both soluble and insoluble proteins are detailed. DEA extracts soluble proteins, such as Aβ isoforms and APP. Formic acid enables extraction of insoluble protein aggregates, such as Aβ isoforms associated with plaques. This procedure produces soluble and insoluble extracts that are amenable to analysis of Aβ species via western blotting and/or enzyme-linked immunosorbent assays (ELISAs), and these results help assess amyloidogenic burden in animals.

Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer's disease.

The age-dependent deposition of amyloid-β peptides, derived from amyloid precursor protein (APP), is a neuropathological hallmark of Alzheimer's disease (AD). Despite age being the greatest risk factor for AD, the molecular mechanisms linking ageing to APP processing are unknown. Here we show that asparagine endopeptidase (AEP), a pH-controlled cysteine proteinase, is activated during ageing and mediates APP proteolytic processing. AEP cleaves APP at N373 and N585 residues, selectively influencing the amyloidogenic fragmentation of APP. AEP is activated in normal mice in an age-dependent manner, and is strongly activated in 5XFAD transgenic mouse model and human AD brains. Deletion of AEP from 5XFAD or APP/PS1 mice decreases senile plaque formation, ameliorates synapse loss, elevates long-term potentiation and protects memory. Blockade of APP cleavage by AEP in mice alleviates pathological and behavioural deficits. Thus, AEP acts as a δ-secretase, contributing to the age-dependent pathogenic mechanisms in AD.

AβPP-Transgenic 2576 Mice Mimic Cell Type-Specific Aspects of Acetyl-CoA-Linked Metabolic Deficits in Alzheimer's Disease.

The pyruvate-derived acetyl-CoA is a principal direct precursor substrate for bulk energy synthesis in the brain. Deficits of pyruvate dehydrogenase in the neocortex are common features of Alzheimer's disease and other age-related encephalopathies in humans. Therefore, amyloid-β overload in brains of diverse transgenic Alzheimer's disease model animals was investigated as one of neurotoxic compounds responsible for pyruvate dehydrogenase inhibition yielding deficits of cholinergic neurotransmission and cognitive functions. Brains of aged, 14-16-month-old Tg2576 mice contained 0.6 μmol/kg levels of amyloid-β1 - 42. Activities of pyruvate dehydrogenase complex, choline acetyltransferase, and several enzymes of acetyl-CoA and energy metabolism were found to be unchanged in both forebrain mitochondria and synaptosomes of Tg2576 mice, indicating preservation of structural integrity at least in cholinergic neuronal cells. However, in transgenic brain synaptosomes, pyruvate utilization, mitochondrial levels, and cytoplasmic acetyl-CoA levels, as well as acetylcholine content and its quantal release, were all found to be decreased by 25-40% . On the contrary, activation of pyruvate utilization was detected and no alterations in acetyl-CoA content and citrate or α-ketoglutarate accumulation were observed in transgenic whole brain mitochondria. These data indicate that amyloid-β evoked deficits in acetyl-CoA are confined to mitochondrial and cytoplasmic compartments of Tg2576 nerve terminals, becoming early primary signals paving the path for further stages of neurodegeneration. On the other hand, acetyl-CoA synthesis in mitochondrial compartments of glial cells seems to be activated despite amyloid-β accumulated in transgenic brains.

Klotho Protects Dopaminergic Neuron Oxidant-Induced Degeneration by Modulating ASK1 and p38 MAPK Signaling Pathways.

Klotho transgenic mice exhibit resistance to oxidative stress as measured by their urinal levels of 8-hydroxy-2-deoxyguanosine, albeit this anti-oxidant defense mechanism has not been locally investigated in the brain. Here, we tested the hypothesis that the reactive oxygen species (ROS)-sensitive apoptosis signal-regulating kinase 1 (ASK1)/p38 MAPK pathway regulates stress levels in the brain of these mice and showed that: 1) the ratio of free ASK1 to thioredoxin (Trx)-bound ASK1 is relatively lower in the transgenic brain whereas the reverse is true for the Klotho knockout mice; 2) the reduced p38 activation level in the transgene corresponds to higher level of ASK1-bound Trx, while the KO mice showed elevated p38 activation and lower level of–bound Trx; and 3) that 14-3-3ζ is hyper phosphorylated (Ser-58) in the transgene which correlated with increased monomer forms. In addition, we evaluated the in vivo robustness of the protection by challenging the brains of Klotho transgenic mice with a neurotoxin, MPTP and analyzed for residual neuron numbers and integrity in the substantia nigra pars compacta. Our results show that Klotho overexpression significantly protects dopaminergic neurons against oxidative damage, partly by modulating p38 MAPK activation level. Our data highlight the importance of ASK1/p38 MAPK pathway in the brain and identify Klotho as a possible anti-oxidant effector.

Cytoplasmic mislocalization of RNA splicing factors and aberrant neuronal gene splicing in TDP-43 transgenic pig brain.

BackgroundTAR DNA-binding protein 43 (TDP-43) is a nuclear protein, but it is redistributed in the neuronal cytoplasm in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Because small transgenic animal models often lack cytoplasmic TDP-43, how the cytoplasmic accumulation of TDP-43 contributes to these diseases remains unclear. The current study is aimed at studying the mechanism of cytoplasmic pathology of TDP-43.ResultsWe established transgenic pigs expressing mutant TDP-43 (M337V). This pig model shows severe phenotypes and early death. We found that transgenic TDP-43 is also distributed in the cytoplasm of neuronal cells in the spinal cord and brain. Transgenic TDP-43 interacts with PSF, an RNA splicing factor that associates with NeuN to regulate neuronal RNA splicing. The interaction of TDP-43, PSF and NeuN causes PSF and NeuN mislocalize into the neuronal cytoplasm in transgenic pigs. Consistently, abnormal PSF-related neuronal RNA splicing is seen in TDP-43 transgenic pigs. The cytoplasmic localization of PSF and NeuN as well as abnormal PSF-related neuronal RNA splicing was also found in ALS patient brains.ConclusionOur findings from a large mammalian model suggest that cytoplasmic mutant TDP-43 could reduce the nuclear function of RNA splicing factors, contributing to neuropathology.

Nicotine mediates expression of genes related to antioxidant capacity and oxidative stress response in HIV-1 transgenic rat brain.

Oxidative stress plays an important role in the progression of HIV-1 infection. Nicotine can either protect neurons from neurodegeneration or induce oxidative stress, depending on its dose and degree of oxidative stress impairment. However, the relationship between nicotine and oxidative stress in the HIV-1-infected individuals remains largely unknown. The purpose of this study was to determine the effect of nicotine on expression of genes related to the glutathione (GSH)-centered antioxidant system and oxidative stress in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of HIV-1 transgenic (HIV-1Tg) and F344 control rats. Adult HIV-1Tg and F344 rats received nicotine (0.4mg/kg, base, s.c.) or saline injections once per day for 27days. At the end of treatment, various brain regions including the NAc and VTA were collected from each rat. Following total RNA extraction and complementary DNA (cDNA) synthesis of each sample, quantitative reverse transcription PCR (RT-PCR) analysis was performed for 43 oxidative-stress-related genes. Compared with F344 control rats, HIV-1Tg rats showed a significant downregulation of genes involved in ATPase and cyctochrome oxidase at the messenger RNA (mRNA) level in both regions. Further, we found a significant downregulation of Gstm5 in the NAc and upregulation of Cox1, Cox3, and Gsta6 in the VTA of HIV-1Tg rats. HIV-1Tg rats showed brain-region-specific responses to chronic nicotine treatment. This response resulted in a change in the expression of genes involved in antioxidant mechanisms including the downregulation of genes such as Atp5h, Calml1, Gpx7, Gstm5, Gsr, and Gsta6 and upregulation of Sod1 in the NAc, as well as downregulation of genes like Cox5a, Gpx4, Gpx6, Gpx7, Gstm5, and Sod1 in the VTA of HIV-1Tg rats. Together, we conclude that chronic nicotine treatment has a dual effect on the antioxidant defense system and oxidative-stress-induced apoptosis signaling in HIV-1Tg rats. These findings suggest that nicotine has a negative effect on response to oxidative stress and antioxidant processes in HIV-1 Tg rat brain, especially in the VTA.

Drugs of Abuse in HIV infection and neurotoxicity.

HIV is a neurotropic virus that enters the brain right after infection. In the brain, HIV replicates in macrophages, microglia and small number of astrocytes (4.7 ± 2.8% in vitro and 8.2 ± 3.9 in vivo) (Eugenin et al., 2011) causing inflammatory and neurotoxic host responses. Severe neurological disorders caused by HIV are collectively known as HIV-associated neurocognitive disorders (HAND). HAND is characterized by development of abnormal reduction of motor speed, concentration, and memory. HAND consists of several clinical forms ranging from asymptomatic neurocognitive impairment (ANI), minor neurocognitive disorder (MND) to the most severe HIV-associated dementia (HAD) (McArthur and Brew, 2010). HIV-encephalitis (HIVE) is the main cause of HAND and the most common neurologic disorder of the brain in HIV-1 infection. HIV-1 exhibits extensive genetic variation worldwide and is categorized into three groups (M, O, and N) and genetically into nine different subtypes (A–K). Of these, clades B and C represent the majority (>86%) of circulating HIV-1 variants (Osmanov et al., 2002). While HIV-1 clade B is predominant in North America, Western Europe, and Australia; clade C is common in Southern and East Africa, India and Nepal (responsible for around half of all HIV infections). HIV-1 clade B has been reported to be more neuropathogenic than clade C (Atluri et al., 2013; Samikkannu et al., 2014). Before the worldwide use of highly antiretroviral therapy (HAART), approximately, 20–30% of individuals with advanced HIV-1 clade B infection showed symptoms of HAD (Gonzalez-Scarano and Martin-Garcia, 2005; Kaul et al., 2005). Although the prevalence of HAD has decreased intensely after the introduction of HAART, 40–50% of HIV positive patients still suffer from HAND (Sacktor et al., 2001; Sacktor, 2002; McArthur, 2004; Antinori et al., 2007; Ellis et al., 2007). In developed countries, about 30% of HIV-positive individuals are intravenous drug abusers, which place them in a higher risk for HAND (Miro et al., 2003; Beyrer et al., 2010). Cocaine and marijuana are the most common drugs of abuse among HIV patients, whereas opioids are abused only by a small number of patients (Kuo et al., 2004; Cook et al., 2007; Korthuis et al., 2008). Overall, several drugs of abuse such as tobacco, stimulants, cannabinoids, opioids and alcohol are found to be consumed among HIV infected individuals, having an effect on synaptic plasticity and development found in the brain (Hauser and Knapp, 2014). Figure ​Figure11 is showing different neurotoxic mechanisms of drugs of abuse in HIV infection which may lead to the impaired neurocognitive functions. Figure 1 Schematic representation of neurotoxic mechanisms of different illicit drugs of abuse in HIV infection. Nicotine and HIV Recently, in nicotine and HIV infected SK-N-MC cells, an up-regulation of HDAC2 was observed (Atluri et al., 2014). HDAC2 overexpression has been reported in depleted memory formation, synaptic plasticity and dendritic spine density (Guan et al., 2009). Nevertheless, use of nicotine in infected patients can have beneficial outcomes on neurological deficits that were HIV-1 induced (Cao et al., 2013). In nicotine injected HIV-1 transgenic rats brain regions, such as in the prefrontal cortex, Wnt/β-catenin signaling has shown improvement by restoring the down-regulation of Axin1, Wnt5a, Wnt7a and the up-regulation of Gnao1 (Cao et al., 2013). This signaling pathway plays an important role in the early development of the nervous system, in which a neuroprotective outcome in adults is established after the activation of this pathway (Nave and Trapp, 2008). These results are important since central demyelination and neurodegeneration have been observed in HIV-1 infected individuals. In the dorsal hippocampus, CREB signaling has also been restored, in which the decreased expression of calcium sensor proteins, Calm3 and Cabp1, was regulated to normal levels. This signaling pathway is significant in neuronal survival and long-term synaptic plasticity. In the dorsal striatum, nicotine has also shown to restore the function of the tricarboxylic acid (TCA) cycle and its related pathways, such as the down-regulation of Idh3B and up-regulation of Ndufs4 that came back to normal levels (Cao et al., 2013).

Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases including Alzheimer’s disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation, and triggers neurodegeneration. AEP is upregulated and active during aging, and is activated in tau P301S transgenic mice and human AD brain, leading to tau truncation in NFTs. Deletion of AEP from tau P301S transgenic mice substantially reduces tau hyperphosphorylation, alleviates the synapse loss and rescues impaired hippocampal synaptic function and the cognitive deficits. Infection of uncleavable tau N255AN368A mutant rescues tau P301S-induced pathological and behavioral defects. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes in neurodegenerative diseases. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

In vivo optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

&lt;em&gt;In Vivo&lt;/em&gt; Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models.

Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc+/SV40Tag+/Tet-on+) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cells were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibrillary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibrillary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibrillary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cells. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

Notch1-Induced Brain Tumor Models the Sonic Hedgehog Subgroup of Human Medulloblastoma

While activation of the Notch pathway is observed in many human cancers, it is unknown whether elevated Notch1 expression is sufficient to initiate tumorigenesis in most tissues. To test the oncogenic potential of Notch1 in solid tumors, we expressed an activated form of Notch1 (N1ICD) in the developing mouse brain. N1ICD;hGFAP-cre mice were viable but developed severe ataxia and seizures, and died by weaning age. Analysis of transgenic embryo brains revealed that N1ICD expression induced p53-dependent apoptosis. When apoptosis was blocked by genetic deletion of p53, 30% to 40% of N1ICD;GFAP-cre;p53(+/-) and N1ICD;GFAP-cre;p53(-/-) mice developed spontaneous medulloblastomas. Interestingly, N1ICD-induced medulloblastomas most closely resembled the sonic hedgehog subgroup of human medulloblastoma at the molecular level. Surprisingly, N1ICD-induced tumors do not maintain high levels of the Notch pathway gene expression, except for Notch2, showing that initiating oncogenic events may not be decipherable by analyzing growing tumors in some cases. In summary, this study shows that Notch1 has an oncogenic potential in the brain when combined with other oncogenic hits, such as p53 loss, and provides a novel mouse model of medulloblastoma. Cancer Res; 73(17); 5381-90. ©2013 AACR.