U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease.

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of ß-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

Neuronal RING finger protein 11 (RNF11) regulates canonical NF-κB signaling.

BACKGROUND: The RING domain-containing protein RING finger protein 11 (RNF11) is a member of the A20 ubiquitin-editing protein complex and modulates peripheral NF-κB signaling. RNF11 is robustly expressed in neurons and colocalizes with a population of α-synuclein-positive Lewy bodies and neurites in Parkinson disease patients. The NF-κB pathway has an important role in the vertebrate nervous system, where the absence of NF-κB activity during development can result in learning and memory deficits, whereas chronic NF-κB activation is associated with persistent neuroinflammation. We examined the functional role of RNF11 with respect to canonical NF-κB signaling in neurons to gain understanding of the tight association of inflammatory pathways, including NF-κB, with the pathogenesis of neurodegenerative diseases. METHODS AND RESULTS: Luciferase assays were employed to assess NF-κB activity under targeted short hairpin RNA (shRNA) knockdown of RNF11 in human neuroblastoma cells and murine primary neurons, which suggested that RNF11 acts as a negative regulator of canonical neuronal NF-κB signaling. These results were further supported by analyses of p65 translocation to the nucleus following depletion of RNF11. Coimmunoprecipitation experiments indicated that RNF11 associates with members of the A20 ubiquitin-editing protein complex in neurons. Site-directed mutagenesis of the myristoylation domain, which is necessary for endosomal targeting of RNF11, altered the impact of RNF11 on NF-κB signaling and abrogated RNF11's association with the A20 ubiquitin-editing protein complex. A partial effect on canonical NF-κB signaling and an association with the A20 ubiquitin-editing protein complex was observed with mutagenesis of the PPxY motif, a proline-rich region involved in Nedd4-like protein interactions. Last, shRNA-mediated reduction of RNF11 in neurons and neuronal cell lines elevated levels of monocyte chemoattractant protein 1 and TNF-α mRNA and proteins, suggesting that NF-κB signaling and associated inflammatory responses are aberrantly regulated in the absence of RNF11. CONCLUSIONS: Our findings support the hypothesis that, in the nervous system, RNF11 negatively regulates canonical NF-κB signaling. Reduced or functionally compromised RNF11 could influence NF-κB-associated neuronal functions, including exaggerated inflammatory responses that may have implications for neurodegenerative disease pathogenesis and progression.

Exogenous seeding of cerebral ß-amyloid deposition in ßAPP-transgenic rats.

Deposition of the amyloid-ß (Aß) peptide in senile plaques and cerebral Aß angiopathy (CAA) can be stimulated in Aß-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aß seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aß itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aß seeding have employed animal models that, as they age, eventually will generate Aß lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aß within the course of its median lifespan (30 months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aß. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aß-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aß.

Aberrant septin 11 is associated with sporadic frontotemporal lobar degeneration.

BACKGROUND: Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches. RESULTS: We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Moreover, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, SEPT11, in detail. Using independent highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved into N-terminal fragments and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of FTLD-U patients. CONCLUSIONS: The proteomic discovery of insoluble SEPT11 accumulation in FTLD-U, along with novel pathological associations, highlights a role for this cytoskeleton-associated protein in the pathogenesis of this complex disorder.

Deletion of M1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo.

Alzheimer's disease (AD) is a progressive neurological disorder that causes dementia and poses a major public health crisis as the population ages. Aberrant processing of the amyloid precursor protein (APP) is strongly implicated as a proximal event in AD pathophysiology, but the neurochemical signals that regulate APP processing in the brain are not completely understood. Activation of muscarinic acetylcholine receptors (mAChRs) has been shown to affect APP processing and AD pathology, but less is known about the roles of specific mAChR subtypes. In this study, we used M(1) mAChR knock-out mice (M(1)KO) to isolate the effects of the M(1) mAChR on APP processing in primary neurons and on the development of amyloid pathology in a transgenic mouse model of AD. We demonstrate that the loss of M(1) mAChRs increases amyloidogenic APP processing in neurons, as evidenced by decreased agonist-regulated shedding of the neuroprotective APP ectodomain APPsalpha and increased production of toxic Abeta peptides. Expression of M(1) mAChRs on the M(1)KO background rescued this phenotype, indicating that M(1) mAChRs are sufficient to modulate nonamyloidogenic APP processing. In APP(Swe/Ind) transgenic mice, the loss of M(1) mAChRs resulted in increased levels of brain Abeta and greater accumulation of amyloid plaque pathology. Analysis of APP metabolites in APP(Swe/Ind) brain tissue indicates that the loss of M(1) mAChRs increases amyloidogenic APP processing. These results indicate that the M(1) mAChR is an important regulator of amyloidogenesis in the brain and provide strong support for targeting the M(1) mAChR as a therapeutic candidate in AD.

DHA diet reduces AD pathology in young APPswe/PS1 Delta E9 transgenic mice: possible gender effects.

Epidemiological and clinical trial findings suggest that consumption of docosahexaenoic acid (DHA) lowers the risk of Alzheimer's disease (AD). We examined the effects of short-term (3 months) DHA enriched diet on plaque deposition and synaptic defects in forebrain of young APPswe/PS1 Delta E9 transgenic (tg) and non-transgenic (ntg) mice. Gas chromatography revealed a significant increase in DHA concomitant with a decrease of arachidonic acid in both brain and liver in mice fed with DHA. Female tg mice consumed relatively more food daily than ntg female mice, independent of diet. Plaque load was significantly reduced in the cortex, ventral hippocampus and striatum of female APPswe/PS1 Delta E9 tg mice on DHA diet compared to female tg mice on control diet. Immunoblot quantitation of the APOE receptor, LR11, which is involved in APP trafficking and A beta production, were unchanged in mice on DHA or control diets. Moreover drebrin levels were significantly increased in the hippocampus of tg mice on the DHA diet. Finally, in vitro DHA treatment prevented amyloid toxicity in cell cultures. Our findings support the concept that increased DHA consumption may play and important role in reducing brain insults in female AD patients.

Loss of LR11/SORLA enhances early pathology in a mouse model of amyloidosis: evidence for a proximal role in Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia, resulting in progressive neuronal death and debilitating damage to brain loci that mediate memory and higher cognitive function. While pathogenic genetic mutations have been implicated in approximately 2% of AD cases, the proximal events that underlie the common, sporadic form of the disease are incompletely understood. Converging lines of evidence from human neuropathology, basic biology, and genetics have implicated loss of the multifunctional receptor LR11 (also known as SORLA and SORL1) in AD pathogenesis. Cell-based studies suggest that LR11 reduces the formation of beta-amyloid (Abeta), the molecule believed to be a primary toxic species in AD. Recently, mutant mice deficient in LR11 were shown to upregulate murine Abeta in mouse brain. In the current study, LR11-deficient mice were crossed with transgenic mice expressing autosomal-dominant human AD genes, presenilin-1 (PS1DeltaE9) and amyloid precursor protein (APPswe). Here, we show that LR11 deficiency in this AD mouse model significantly increases Abeta levels and exacerbates early amyloid pathology in brain, causing a forward shift in disease onset that is LR11 gene dose-dependent. Loss of LR11 increases the processing of the APP holo-molecule into alpha-, beta-, and gamma-secretase derived metabolites. We propose that LR11 regulates APP processing and Abeta accumulation in vivo and is of proximal importance to the cascade of pathological amyloidosis. The results of the current study support the hypothesis that control of LR11 expression may exert critical effects on Alzheimer's disease susceptibility in humans.

Fas-associated factor 1 and Parkinson's disease.

Fas-associated factor 1 or FAF1 is a Fas-binding protein implicated in apoptosis. FAF1 is the product of a gene at PARK 10 locus on chromosome 1p32, a locus associated with late-onset PD [Hicks, A.A., Petursson, H., Jonsson, T., Stefansson, H., Johannsdottir, H.S., Sainz, J., Frigge, M.L.et al., 2002. A susceptibility gene for late-onset idiopathic Parkinson's disease. Ann Neurol. 52, 549-555.]. In the present study we investigated the role of FAF1 in cell death and in Parkinson's disease (PD) pathogenesis. FAF1 levels were significantly increased in frontal cortex of PD as well as in PD cases with Alzheimer's disease (AD) pathology compared to control cases. Changes in FAF1 expression were specific to PD-related alpha-synuclein pathology and nigral cell loss. In addition, PD-related insults including, mitochondrial complex I inhibition, oxidative stress, and increased alpha-synuclein expression specifically increased endogenous FAF1 expression in vitro. Increased FAF1 levels induced cell death and significantly potentiated toxic effects of PD-related stressors including, oxidative stress, mitochondrial complex I inhibition and proteasomal inhibition. These studies, together with previous genetic linkage studies, highlight the potential significance of FAF1 in pathogenesis of idiopathic PD.

Development of transgenic rats producing human beta-amyloid precursor protein as a model for Alzheimer's disease: transgene and endogenous APP genes are regulated tissue-specifically.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the beta-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. RESULTS: Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Abeta)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Abeta42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. CONCLUSION: This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

Increased sensitivity to light-induced damage in a mouse model of autosomal dominant retinal disease.

PURPOSE: To describe a sensitivity to light-induced damage associated with expression of a T17M mutant human rhodopsin (hT17M) transgene in mice, with the goal of minimizing retinal injury during the subretinal delivery of rAAV-mediated gene therapy. METHODS: Mice were bred to express the hT17M rhodopsin transgene in a line that was hemizygous null for wild-type mouse rhodopsin (mrho(+/-)), and the eyes of transgenic mice and nontransgenic littermates were exposed for 2.5 minutes to unilateral illumination with fiber-optic light ranging from 5,000 to 10,000 lux. Funduscopic images were made with a handheld camera (Genesis; Kowa Company, Ltd., Tokyo, Japan). Full-field scotopic electroretinographic analysis (ERG) was performed to measure loss of retinal function. Morphometry in the light microscope was used to measure loss of rod photoreceptors. TUNEL staining and a nucleosome release assay were used to measure levels of apoptosis in retinal specimens. RESULTS: mrho(+/-);hT17M mice exhibited a sensitivity to light-induced damage that caused severe loss of a- and b-wave ERG responses. hT17M transgenic mice on the mrho(+/+) background were equally sensitive to light-induced damage. Histologic analysis showed a concomitant loss of photoreceptors and TUNEL labeling of fragmented DNA in rod photoreceptor cells, demonstrating that the damage occurred via an apoptotic pathway. Nontransgenic littermate mice were not affected by this exposure to light. Mice expressing an hP23H mutant human rhodopsin transgene were minimally sensitive to light-induced damage at these intensities, in comparison to hT17M mice. Treating the hT17M mice with an equivalent regimen of exposure to red light was less damaging to the retina, as measured by ERG and histology. CONCLUSIONS: Expression of a human hT17M mutant rhodopsin transgene in mice is associated with photoreceptor apoptosis in response to moderate exposure to light. This phenotype was not observed in nontransgenic littermates or in mice expressing an hP23H mutant human rhodopsin transgene. The results suggest that elimination of the glycosylation site at N15 is associated with increased sensitivity to light-induced damage.

The lipoprotein receptor LR11 regulates amyloid beta production and amyloid precursor protein traffic in endosomal compartments.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and neuropathological changes, including the deposition of amyloid beta (Abeta) in senile plaques. The mechanisms causing the disease and Abeta accumulation are not well understood, but important genetic associations with apolipoprotein E genotype and involvement of lipoprotein receptors have become apparent. LR11 (also known as SorLA), a member of the low-density lipoprotein receptor family, has been identified previously as an altered transcript in microarray analyses of samples from human AD cases. Here, we show neuronal expression of the lipoprotein receptor LR11 in control brain in regions vulnerable to AD neuropathology and marked reduction of LR11 expression in these regions in AD brains before cell death. Overexpression of LR11 drastically reduces levels of extracellular Abeta and also lowers levels of total cellular amyloid precursor protein (APP). LR11 colocalizes with APP and regulates its trafficking in endocytic compartments, which are important intracellular sites for APP processing and Abeta generation. Endogenous LR11 localizes to neuronal multivesicular bodies in both rat and human brain. The robust correlation between reduced LR11 expression and AD neuropathology and its potent effects on extracellular Abeta levels suggest that this neuronal lipoprotein receptor could play an important role in AD pathogenesis.

Design and validation of therapeutic hammerhead ribozymes for autosomal dominant diseases.

Hammerhead ribozymes are small, catalytic RNAs that can be designed to effectively inhibit gene expression in an allele-specific manner. It is the high level of sequence discrimination, coupled with the minimal cleavage-site requirements of hammerhead ribozymes, that makes these catalytic RNAs so amenable for use as therapeutic agents for autosomal dominant diseases. Here, we present a detailed set of protocols for the design and validation of hammerhead ribozymes for the treatment of autosomal dominant disease, with specific examples of hammerhead ribozymes targeted against human P23H rod opsin mRNA, a major cause of dominant retinitis pigmentosa.