Colleen L. Forster

Non-ATG-initiated translation directed by microsatellite expansions

Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.

Age-Dependent Neurofibrillary Tangle Formation, Neuron Loss, and Memory Impairment in a Mouse Model of Human Tauopathy (P301L)

Here, we describe the generation of a novel transgenic mouse model of human tauopathy. The rTg(tauP301L)4510 mouse expresses the P301L mutation in tau (4R0N) associated with frontotemporal dementia and parkinsonism linked to chromosome 17. Transgene expression was driven by a forebrain-specific Ca2+ calmodulin kinase II promoter system resulting in high levels of expression in the hippocampus and neocortex. Importantly, transgene expression in this model is induced via the tetracycline-operon responsive element and is suppressed after treatment with doxycycline. Continued transgene expression in rTg(tauP301L)4510 mice results in age-dependent development of many salient characteristics of hereditary human dementia. From an early age, immunohistochemical studies demonstrated abnormal biochemical processing of tau and the presence of pathological conformation- and phosphorylation-dependent epitopes. Neurofibrillary tangle (NFT) pathology was first observed in the neocortex and progressed into the hippocampus and limbic structures with increasing age. Consistent with the formation of NFTs, immunoblots indicated an age-dependent transition of accumulating tau species from Sarkosyl soluble 55 kDa to insoluble hyperphosphorylated 64 kDa. Ultrastructural analysis revealed the presence of straight tau filaments. Furthermore, the effects of tauP301L expression on spatial reference memory were longitudinally tested using the Morris water maze. Compared with nontransgenic age-matched control littermates, rTg(tauP301L)4510 mice developed significant cognitive impairments from 4 months of age. Memory deficits were accompanied by gross forebrain atrophy and a prominent loss of neurons, most strikingly in hippocampal subdivision CA1. Collectively, these data describe a novel transgenic mouse that closely mimics human tauopathy and may represent an important model for the future study of tau-related neurodegenerative disease.

Inducible nitric oxide synthase expression in human cerebral infarcts

Abstract The inducible or “immunological” isoform of nitric oxide synthase (iNOS) is induced in many cell types by inflammatory stimuli and synthesizes toxic amounts of NO. In rodent models of focal cerebral ischemia, iNOS is expressed in neutrophils invading the injured brain and in local blood vessels. Studies with iNOS inhibitors and iNOS null mice indicate that NO produced by iNOS contributes to ischemic brain injury. In the present study, we sought to determine whether iNOS is also expressed in the human brain after ischemic stroke. Studies were conducted using immunohistochemistry on autopsy brains with neuropathological evidence of acute cerebral infarction. iNOS immunoreactivity was observed in neutrophils infiltrating the ischemic brain and in blood vessels within the ischemic territory. iNOS-positive cells also were immunoreactive for nitrotyrosine, reflecting protein nitration by NO-derived peroxynitrite and nitrites. iNOS or nitrotyrosine immunoreactivity was not detected outside the region of the infarct. These observations provide evidence that iNOS is expressed in the human brain after ischemic infarction and support the hypothesis that iNOS inhibitors may be useful in the treatment of ischemic stroke in humans.

Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia

Abstract The prostaglandin synthesizing enzyme cyclooxygenase-2 (COX-2) is up-regulated in the brain of rodents during cerebral ischemia and contributes to ischemic brain injury. This study sought to determine whether COX-2 is also up-regulated in the human brain in the acute stages of cerebral ischemic infarction. Paraffin-embedded sections from patients who died 1–2 days following infarction in the middle cerebral artery territory were processed for COX-2 immunohistochemistry. COX-2 immunoreactivity was observed in infiltrating neutrophils, in vascular cells and in neurons located at the border of the infarct. The data suggest that COX-2 up-regulation is also relevant to cerebral ischemia in humans and raise the possibility that COX-2 reaction products participate in the mechanisms of ischemic injury also in the human brain.

Simian Immunodeficiency Virus—Induced Intestinal Cell Apoptosis Is the Underlying Mechanism of the Regenerative Enteropathy of Early Infection

The enteropathic manifestations of the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV) in late infection are usually due to infection by other microbes, but in early infection the viruses themselves cause an enteropathy by heretofore undetermined mechanisms. Here we report that SIV induces massive apoptosis of intestinal epithelial cells lining the small and large bowel, thus identifying apoptosis as the driving force behind the regenerative pathology of early infection. We found that apoptosis of gut epithelium paralleled the previously documented apoptosis and massive depletion of CD4 T cells in gut lamina propria, triggered by established mechanisms of gut epithelial cell apoptosis and, at peak, possibly by virus interactions with GPR15/Bob, an intestinal epithelial cell-associated alternative coreceptor for SIV and HIV-1. Apoptosis in early SIV infection is thus the common theme of the pathological processes that quickly afflict the innate as well as adaptive arms of the gut immune system.

Involvement of β-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synaptic plasticity

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-β protein (Aβ), which forms amyloid plaques in Alzheimers disease (AD), secreted APPα (sAPPα) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The β-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Aβ, AICD, and secreted APPβ. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925–937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

A Sleeping Beauty forward genetic screen identifies new genes and pathways driving osteosarcoma development and metastasis

Osteosarcomas are sarcomas of the bone, derived from osteoblasts or their precursors, with a high propensity to metastasize. Osteosarcoma is associated with massive genomic instability, making it problematic to identify driver genes using human tumors or prototypical mouse models, many of which involve loss of Trp53 function. To identify the genes driving osteosarcoma development and metastasis, we performed a Sleeping Beauty (SB) transposon-based forward genetic screen in mice with and without somatic loss of Trp53. Common insertion site (CIS) analysis of 119 primary tumors and 134 metastatic nodules identified 232 sites associated with osteosarcoma development and 43 sites associated with metastasis, respectively. Analysis of CIS-associated genes identified numerous known and new osteosarcoma-associated genes enriched in the ErbB, PI3K-AKT-mTOR and MAPK signaling pathways. Lastly, we identified several oncogenes involved in axon guidance, including Sema4d and Sema6d, which we functionally validated as oncogenes in human osteosarcoma.

Quaternary Structure Defines a Large Class of Amyloid-β Oligomers Neutralized by Sequestration

The accumulation of amyloid-β (Aβ) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimers disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aβ oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aβ oligomers in vivo.

Canonical Wnt/β-catenin Signaling Drives Human Schwann Cell Transformation, Progression, and Tumor Maintenance

Genetic changes required for the formation and progression of human Schwann cell tumors remain elusive. Using a Sleeping Beauty forward genetic screen, we identified several genes involved in canonical Wnt signaling as potential drivers of benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). In human neurofibromas and MPNSTs, activation of Wnt signaling increased with tumor grade and was associated with downregulation of β-catenin destruction complex members or overexpression of a ligand that potentiates Wnt signaling, R-spondin 2 (RSPO2). Induction of Wnt signaling was sufficient to induce transformed properties in immortalized human Schwann cells, and downregulation of this pathway was sufficient to reduce the tumorigenic phenotype of human MPNST cell lines. Small-molecule inhibition of Wnt signaling effectively reduced the viability of MPNST cell lines and synergistically induced apoptosis when combined with an mTOR inhibitor, RAD-001, suggesting that Wnt inhibition represents a novel target for therapeutic intervention in Schwann cell tumors.

Amyloid Plaque and Neurofibrillary Tangle Pathology in a Regulatable Mouse Model of Alzheimer's Disease

Transgenic mouse models that independently express mutations in amyloid precursor protein (APP) and tau have proven useful for the study of the neurological consequences of amyloid-beta (Abeta) plaque and neurofibrillary tangle pathologies. Studies using these mice have yielded essential discoveries with regard to specific aspects of neuronal dysfunction and degeneration that characterize the brain during Alzheimers disease (AD) and other age-dependent tauopathies. Most recent transgenic studies have focused on the creation of regulatable models that allow the temporal control of transgene expression. To study a more complete model of AD pathology, we designed a new regulatable transgenic mouse that harbors both APP and tau transgenes. Here, we present a novel transgenic mouse model, rTg3696AB, which expresses human APP(NLI) and tau(P301L) driven by the CaMKII promoter system. Subsequent generation of Abeta and 4R0N tau in the brain resulted in the development of three neuropathological features of AD: Abeta plaques, neurofibrillary tangles, and neurodegeneration. Importantly, transgene expression in these mice is regulatable, permitting temporal control of gene expression and the investigation of transgene suppression.