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The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer's disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in ∼50% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography-tandem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 ± 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer's disease.

The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.

Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly. Because the pathological changes underlying this disease can begin decades prior to the onset of cognitive impairment, identifying the earliest events in the AD pathological cascade has critical implications for both the diagnosis and treatment of this disease. We previously reported that compared to autopsy confirmed healthy control brain, expression of LR11 (or SorLA) is markedly reduced in AD brain as well as in a subset of people with mild cognitive impairment (MCI), a prodromal clinical stage of AD. Recent studies of the LR11 gene SORL1 have suggested that the association between SORL1 single nucleotide polymorphisms (SNPs) and AD risk may not be universal. Therefore, we sought to confirm our earlier findings in a population chosen solely based on clinical criteria, as in most genetic studies. Quantitative immunohistochemistry was used to measure LR11 expression in 43 cases from the Religious Orders Study that were chosen based on a final pre-mortem clinical diagnosis of MCI, mild/moderate AD or no cognitive impairment (NCI). LR11 expression was highly variable in all three diagnostic groups, with no significant group differences. Low LR11 cases were identified using the lowest tertile of LR11 expression observed across all cases as a threshold. Contrary to previous reports, low LR11 expression was found in only 29% of AD cases. A similar proportion of both the MCI and NCI cases also displayed low LR11 expression. AD-associated lesions were present in the majority of cases regardless of diagnostic group, although we found no association between LR11 levels and pathological variables. These findings suggest that the relationship between LR11 expression and the development of AD may be more complicated than originally believed.

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.

M(1) muscarinic acetylcholine receptors (mAChRs) may represent a viable target for treatment of disorders involving impaired cognitive function. However, a major limitation to testing this hypothesis has been a lack of highly selective ligands for individual mAChR subtypes. We now report the rigorous molecular characterization of a novel compound, benzylquinolone carboxylic acid (BQCA), which acts as a potent, highly selective positive allosteric modulator (PAM) of the rat M(1) receptor. This compound does not directly activate the receptor, but acts at an allosteric site to increase functional responses to orthosteric agonists. Radioligand binding studies revealed that BQCA increases M(1) receptor affinity for acetylcholine. We found that activation of the M(1) receptor by BQCA induces a robust inward current and increases spontaneous EPSCs in medial prefrontal cortex (mPFC) pyramidal cells, effects which are absent in acute slices from M(1) receptor knock-out mice. Furthermore, to determine the effect of BQCA on intact and functioning brain circuits, multiple single-unit recordings were obtained from the mPFC of rats that showed BQCA increases firing of mPFC pyramidal cells in vivo. BQCA also restored discrimination reversal learning in a transgenic mouse model of Alzheimer's disease and was found to regulate non-amyloidogenic APP processing in vitro, suggesting that M(1) receptor PAMs have the potential to provide both symptomatic and disease modifying effects in Alzheimer's disease patients. Together, these studies provide compelling evidence that M(1) receptor activation induces a dramatic excitation of PFC neurons and suggest that selectively activating the M(1) mAChR subtype may ameliorate impairments in cognitive function.

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.

Objective: LR11 (aka sorLA) is a multifunctional neuronal receptor that binds apolipoprotein E and interacts with amyloid precursor protein to regulate amyloidogenesis. Reduced expression of LR11, as occurs in the brains of individuals with Alzheimer's disease (AD), increases amyloidogenesis, and variants in the gene that encodes LR11, SORL1, have recently been linked to risk for late-onset AD. In this study, we sought to determine whether reduced expression of LR11 occurs early in the disease process and whether protein levels in cortical neurons are associated with clinical and pathological changes in mild cognitive impairment (MCI), a condition that may represent prodromal AD., Methods: A novel quantitative immunohistochemical approach was used to measure LR11 levels in brain tissue collected from subjects diagnosed antemortem with either no cognitive impairment, MCI, or AD from the Rush University Religious Orders Study., Results: LR11 levels in MCI were intermediate between no cognitive impairment and AD. LR11 expression was heterogeneous in MCI, forming low- and high-level LR11 subgroups. MCI subjects with low LR11 were significantly more cognitively impaired than the high LR11 subjects. We also found a significant correlation between cognitive performance and LR11 levels across all clinical groups examined. There was no association between LR11 and plaque and tangle pathology., Interpretation: Neuronal LR11 levels are reduced in prodomal AD. The correlation between LR11 expression and cognitive performance indicates that reduced LR11 levels reflect disease severity and may predict progression to AD in a subgroup of individuals with MCI.

The PARK10 locus is associated with idiopathic Parkinson disease (PD), but the responsible gene remains to be identified. Genes associated with familial PD, as well as biochemical evidence from sporadic PD and animal models, have implicated components of the ubiquitin-proteasome system in PD pathogenesis. One attractive candidate gene at the PARK10 locus is RING-Finger Protein 11 (RNF11), the deduced amino acid sequence of which predicts a RING-H2 domain common to E3 ubiquitin ligases such as parkin. To facilitate understanding of this protein and its possible role in PD, we characterized the expression and localization of RNF11 in brain. We detected RNF11 transcript and protein and provided the first direct evidence that RNF11 is expressed in brain. Immunohistochemical analysis of RNF11 protein in rat and human brain, using 2 different antibodies, corroborated the mRNA findings. Both antibodies show that RNF11 is restricted to neurons and excluded from white matter. Moreover, RNF11 is expressed by vulnerable neurons of the substantia nigra and sequestered into Lewy bodies in brains of patients with idiopathic PD. Collectively, these findings identify RNF11 as a strong candidate gene at the PARK10 locus and highlight its potential significance in the development of the common form of PD.

Presenilin-1 (PS1), which is linked to familial Alzheimer's disease, participates in the proteolytic processing of Notch and amyloid-beta precursor protein (APP) by an unknown mechanism. Reports of PS1 localization to the endoplasmic reticulum (ER) and Golgi apparatus have focused attention on the early biosynthetic pathway as the site of PS1 function. However, it is unclear how Notch cleavage and APP processing events which occur at or near the cell surface are influenced by PS1. In contrast to some earlier studies, examination of endogenously expressed PS1 in PC12 cells by subcellular fractionation and immunofluorescence microscopy revealed a distribution distinct from that of ER and Golgi markers. Rather, PS1 colocalized with transferrin receptor, a marker for early endosomes. In addition, electron microscopic examination of intact vesicles immunoisolated with PS1 antibodies allowed visualization of endocytic tracer in endosomes. These findings identify an early endosomal pool of PS1 and suggest alternative mechanisms for PS1 interactions with APP and Notch., (Copyright 2000 Academic Press.)

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

Several synaptic vesicle proteins including synaptophysin and p65/synaptotagmin are expressed by the pheochromocytoma cell line PC12. Stimulation of these cells with nerve growth factor for 7 days induces morphologic neuronotypic differentiation, but the levels of synaptophysin are markedly reduced. Stimulation with cyclic AMP analogs also produces neuronotypic differentiation of PC12 cells, and the degree of morphologic differentiation induced by these agents parallels their ability to effect reduction in synaptophysin levels. By contrast, levels of p65/synaptotagmin are increased following neuronotypic differentiation. The contrasting effects of neuronotypic differentiation on levels of synaptophysin and p65/synaptotagmin indicate potential differences in the regulation of these proteins in PC12 cells. Immunocytochemical labeling of undifferentiated PC12 cells reveals concentrations of synaptophysin in the perinuclear region. After neuronotypic differentiation, there is reduction in perinuclear labeling and concentration of label in swellings along PC12 cell processes. At the ultrastructural level, synaptophysin labeling is found on similar organelles in both undifferentiated and nerve growth factor-stimulated PC12 cells. Although the highest labeling densities were seen on small clear vesicles, specific labeling was also seen on dense core vesicles. The presence of synaptophysin on both small clear vesicles and dense core vesicles indicates potential functional similarities in these vesicle types. The changes in the levels and immunocytochemical distribution of synaptophysin after neuronotypic differentiation suggest possible functional heterogeneity among morphologically similar populations of small clear vesicles.

The growth-associated protein GAP-43 (B-50, F1, pp46), has been found in elongating axons during development and regeneration, and has also been associated with synaptic plasticity in mature neurons. We have examined the loss of GAP-43 labelling from cerebellar granule cells with immunocytochemical localization of a polyclonal antibody to GAP-43. One day after plating, the plasma membrane of cell bodies, neurites and growth cones were all labelled with anti-GAP-43. By 10 days, most of the cell body labelling was lost, and by 20 days the neuritic and growth cone labelling was greatly reduced. Beginning at six days, anti-GAP-43 labelling of growth cones, which was initially uniform, became clustered. When growth cones were double-labelled with antibodies to GAP-43 and the synaptic vesicle protein, p65, inverse changes in the distribution of label was observed. While growth cone labelling with anti-p65 increased from three to 20 days in culture, GAP-43 label began to be lost from some growth cones by six days and showed continuing decline through 20 days. For individual growth cones, the loss of GAP-43 appeared to parallel the accumulation of p65, and first growth cones to lose GAP-43 appeared to be the first to accumulate p65 label. When cultures were grown on a substrate of basement membrane material, the time frames of neuritic outgrowth, loss of GAP-43 labelling, and increase in p65 labelling were all accelerated. At five days, labelling for GAP-43 was weak and labelling for p65 was strong, in a pattern comparable to that seen in older cultures on a polylysine substrate. These results suggest several conclusions concerning the expression and loss of GAP-43 in cultured cerebellar granule neurons. First, GAP-43 label is initially distributed in all parts of these cells. With increasing time in culture the label is first lost from cell bodies and later from neurites and growth cones. Second, the loss of GAP-43 label from growth cones is correlated with the appearance of the synaptic vesicle protein p65. Finally, in vitro developmental changes in the loss of GAP-43 can be altered by changing the growth substrate.