Corrections

CELL BIOLOGY. For the article ‘‘Binding of internalized receptors to follows: ‘‘(A) Indirect immunofluorescence staining of ARPE-19 the PDZ domain of GIPC͞synectin recruits VI to endocytic cells expressing GFP-MegTmT or GFP-MegTmT⌬PDZ stained vesicles,’’ by Samia N. Naccache, Tama Hasson, and Arie Horowitz, for myo6 or synectin. The second and fourth rows are enlargements which appeared in issue 34, August 22, 2006, of Proc Natl Acad Sci of the boxed regions in the first and third rows. Filled arrows USA (103:12735–12740; first published August 14, 2006; 10.1073͞ indicate collocation of GFP constructs with myo6 or with synectin; pnas.0605317103), the authors note that in Fig. 2, the ‘‘myosin VI’’ open arrows indicate the absence of collocation. (Scale bars: 10 ␮m; labels should be replaced with ‘‘’’ in D and with ‘‘R-Tfn’’ in enlarged images, 2.5 ␮m.)’’ The figure and its corrected legend E. The corrected figure and its legend appear below. In addition, the appear below. These errors do not affect the conclusions of the portion of the Fig. 5 legend describing A should have read as article.

Fig. 2. The N-terminal and PDZ domains of synectin are required for UCV binding. (A) Schematic of GFP-, YFP-, and CFP-fused synectin constructs. Residue numbers at the ends of the N-terminal (N), PDZ (P), and C-terminal (C) Fig. 5. Synectin binding to the PBM of megalin is required for synectin and domains are shown above the YFP-synectin construct. (B–F) ARPE-19 cells myo6 recruitment to UCV. (A) Indirect immunofluorescence staining of expressing VFP-fused synectin constructs (green). Boxed areas at Left are ARPE-19 cells expressing GFP-MegTmT or GFP-MegTmT⌬PDZ stained for myo6 enlarged at Right. Furthermost Right is an overlay of the two magnified fields or synectin. The second and fourth rows are enlargements of the boxed to its left. Vesicles showing collocation are indicated by arrows. (B) VFP- regions in the first and third rows. Filled arrows indicate collocation of GFP synectin-expressing cells stained for myo6 (rabbit-anti myo6, red) revealed constructs with myo6 or with synectin; open arrows indicate the absence of significant collocation. (C) The PDZ domain (VFP-syn-P, green) was not re- collocation. (Scale bars: 10 ␮m; enlarged images, 2.5 ␮m.) (B) Quantification of cruited to UCV, and its expression did not interfere with myo6 targeting to the collocation of GFP-MegTmT-containing (black bars) or GFP- vesicles in ARPE-19 cells (anti-myo6, red). (D) VFP-syn-P collocated with focal MegTmT⌬PDZ-containing (white bars) vesicles with endogenous synectin adhesion marker vinculin (red). (E) VFP-syn-NP targeted to peripheral vesicles (syn), myo6 (myo6), AP-2, and R-Tfn along the cell periphery. A total of 150 internally labeled with R-Tfn (red) after 2-min pulse–chase uptake. (F) Expres- vesicles were counted from at least three cells. sion of VFP-syn-NP (green) prevented myo6 (red) recruitment to UCV. (Scale bars: 10 ␮m; enlarged images, 2.5 ␮m.) www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607415103

15272 ͉ PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 www.pnas.org Downloaded by guest on September 28, 2021 BIOCHEMISTRY. For the article ‘‘Genomewide demarcation of IMMUNOLOGY. For the article ‘‘Nonobese diabetic mice express RNA polymerase II transcription units revealed by physical aspects of both type 1 and type 2 diabetes,’’ by Rodolfo Jose´ fractionation of chromatin,’’ by Peter L. Nagy, Michael L. Chaparro, Yves Konigshofer, Georg F. Beilhack, Judith A. Cleary, Patrick O. Brown, and Jason D. Lieb, which appeared in Shizuru, Hugh O. McDevitt, and Yueh-hsiu Chien, which ap- issue 11, May 27, 2003, of Proc Natl Acad Sci USA (100:6364– peared in issue 33, August 15, 2006, of Proc Natl Acad Sci USA 6369; first published May 15, 2003; 10.1073͞pnas.1131966100), (103:12475–12480; first published August 8, 2006; 10.1073͞pnas. the authors note that the data discussed in this publication have 0604317103), the authors note that Georg F. Beilhack should be been deposited in the Expression Omnibus (GEO) data- credited for performing research. The corrected author foot- base, www.ncbi.nlm.nih.gov͞geo (accession no. GSE5662). note, which appears online only, is shown below.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607800103 Author contributions: R.J.C. and Y.-h.C. designed research; R.J.C. and G.F.B. performed research; G.F.B. and J.A.S. con- tributed NOD mice and analytic tools; R.J.C., Y.K., and Y.-h.C. PSYCHOLOGY. For the article ‘‘Transcranial magnetic stimulation analyzed data; and R.J.C., Y.K., H.O.M., and Y.-h.C. wrote the of the visual cortex induces somatotopically organized qualia in paper. blind subjects,’’ by Ron Kupers, Arnaud Fumal, Alain Maertens ͞ ͞ ͞ ͞ de Noordhout, Albert Gjedde, Jean Schoenen, and Maurice www.pnas.org cgi doi 10.1073 pnas.0607192103 Ptito, which appeared in issue 35, August 29, 2006, of Proc Natl Acad Sci USA (103:13256–13260; first published August 17, 2006; 10.1073͞pnas.0602925103), the authors note that some MEDICAL SCIENCES. For the article ‘‘NO synthase 2 (NOS2) dele- portions of the author contributions footnote, which appears tion promotes multiple pathologies in a mouse model of Alz- online only, should be revised to reflect the following. Ron heimer’s disease,’’ by C. A. Colton, M. P. Vitek, D. A. Wink, Q. Kupers, Arnaud Fumal, and Maurice Ptito are credited with Xu, V. Cantillana, M. L. Previti, W. E. Van Nostrand, B. designing and performing the research and analyzing the data. Weinberg, and H. Dawson, which appeared in issue 34, August 22, 2006, of Proc Natl Acad Sci USA (103:12867–12872; first Jean Schoenen, Albert Gjedde, and Alain Maertens de Noord- ͞ hout contributed new reagents͞analytic tools. The corrected published August 14, 2006; 10.1073 pnas.0601075103), the au- author contributions footnote is shown below. The online ver- thor name B. Weinberg should have appeared as J. B. Weinberg. sion has been corrected. The corrected author line appears below. The online version has been corrected. Author contributions: R.K., A.F., and M.P. designed research; R.K., A.F., and M.P. performed research; J.S., A.G., and C. A. Colton, M. P. Vitek, D. A. Wink, Q. Xu, V. Cantillana, A.M.d.N. contributed new reagents͞analytic tools; R.K., A.F., M. L. Previti, W. E. Van Nostrand, J. B. Weinberg, and M.P. analyzed data; and R.K. and M.P. wrote the paper. and H. Dawson

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607806103 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607808103 CORRECTIONS

PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15273 Downloaded by guest on September 28, 2021 NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer’s disease

C. A. Colton*†‡, M. P. Vitek*†, D. A. Wink§, Q. Xu*, V. Cantillana*, M. L. Previti¶, W. E. Van Nostrand¶, J. B. Weinbergʈ, and H. Dawson*

*Division of Neurology, and ʈDepartment of Immunology, Duke University Medical Center, Durham, NC 27710; §Radiation Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and ¶Department of Medicine, Stony Brook University, Stony Brook, NY 11794

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved July 11, 2006 (received for review February 9, 2006) Alzheimer’s disease is characterized by two primary pathological brains of mice expressing the APP Swedish mutation (APPsw) features: amyloid plaques and neurofibrillary tangles. The inter- on a NOS2 knockout background strongly indicates that NO may connection between amyloid and tau aggregates is of intense be protective in AD. interest, but mouse models have yet to reveal a direct interrela- tionship. We now show that NO may be a key factor that connects Results amyloid and tau pathologies. Genetic removal of NO synthase 2 in Mice expressing APPsw on a NOS2 null background were mice expressing mutated amyloid precursor results in assessed for the presence of NOS2 mRNA and brain NOS pathological hyperphosphorylation of mouse tau, its redistribution activity. mRNA for NOS2 was observed in WT and APPsw to the somatodendritic compartment in cortical and hippocampal control littermates, but was not found in APPsw͞NOS2Ϫ/Ϫ or neurons, and aggregate formation. Lack of NO synthase 2 in the NOS2Ϫ/Ϫ brains (Fig. 1A). To determine whether NOS activity amyloid precursor protein Swedish mutant mouse increased insol- fell when NOS2 was deleted in APPsw͞NOS2Ϫ/Ϫ mice, calcium- uble ␤-amyloid peptide levels, neuronal degeneration, caspase-3 independent NOS activity was measured in brain lysates from activation, and tau cleavage, suggesting that NO acts at a junction APPsw͞NOS2Ϫ/Ϫ and APPsw mice by using the arginine-to- point between ␤-amyloid peptides, caspase activation, and tau citrulline conversion assay. A significant decrease in activity was aggregation. observed in the APPsw͞NOS2Ϫ/Ϫ lysates compared with APPsw

alone (Fig. 1B), indicating that NOS activity and, most likely, NO MEDICAL SCIENCES amyloid ͉ chronic neurodegeneration ͉ inducible nitric oxide synthase ͉ production was reduced in our bigenic mouse. Quantitative nitric oxide ͉ tau RT-PCR was used to detect compensatory changes in NOS1 and NOS3. NOS1 mRNA fell by 0.64 Ϯ 0.03-fold (n ϭ 5), whereas n addition to neurodegeneration, Alzheimer’s disease (AD) NOS3 mRNA increased by 1.48 Ϯ 0.13-fold (n ϭ 5) compared Ibrain pathology includes insoluble amyloid deposits and ac- with WT littermates. These changes closely mimicked values Ϫ Ϫ cumulation of abnormally phosphorylated and aggregated forms observed in the NOS2 / mouse (0.63 Ϯ 0.04 for NOS1 and of tau, a binding protein. Attempts to recreate the 1.52 Ϯ 0.23 for NOS3). These data demonstrate that the complete spectrum of AD pathologies in mouse models have had compensatory changes in NOS1 and NOS3 are characteristic of Ϫ Ϫ mixed success. Transgenics that overexpress mutated forms of the NOS2 / deletion. amyloid precursor protein (APP) display amyloid plaques, but The presence of abnormally phosphorylated tau was detected Ϫ Ϫ fail to demonstrate tau pathology that is fully reminiscent of AD in APPsw͞NOS2 / mice (n ϭ 5) by using immunocytochem- (1–5). Both amyloid and tau pathologies are observed in double͞ istry on brain sections with AT8, CP13, and AT180 antibodies to triple transgenic mouse models that express mutated human tau specific, disease-associated phosphorylation sites in and mutated APP, in the presence or absence of mutated (14–16). Immunopositive staining for hyperphosphorylated tau presenilin-1 (6, 7). Although mutated human tau isoforms are was observed in the somatodendritic compartments of numerous observed in dementia, they are typically associated with frontal neurons in the hippocampus (Fig. 2 A and F), globus pallidus Ϫ Ϫ temporal dementias, whereas tau aggregates in AD are com- (Fig. 2B), and frontal cortex (Fig. 2C) in APPsw͞NOS2 / brain. posed of normal tau (8). Capsoni et al. (9) developed a mouse Both AT8 and CP13 produced similar patterns of staining in Ϫ Ϫ expressing recombinant antibodies that neutralizes nerve growth APPsw͞NOS2 / mice, whereas AT8͞CP13 immunostaining Ϫ Ϫ factor and displays nonmutated tau pathology and amyloid was not observed in cortical sections from mNOS2 / littermates plaques. This model implies a major role for nerve growth factor (Fig. 2D) or hippocampal sections from APPsw littermates (Fig. signaling in the pathophysiology of AD, but is associated with the 2E). Immunopositive phospho-tau was also seen in apical den- uncommon presence of brain antibodies. We now report the drites, and intracellular aggregate–like structures were observed Ϫ Ϫ induction of somatodendritic tau pathology in cortical and in some neurons from the APPsw͞NOS2 / brains (Fig. 2B, hippocampal neurons in a well established mouse model of AD arrows). that expresses the Swedish familial AD double mutation K670N- A similar immunostaining pattern was observed in brain M671L in APP (Tg2576) and that lacks a functional NO synthase sections with the AT180 antibody to phosphorylated Ser-231 (NOS) 2 gene. (Fig. 2 G–I). Phospho-tau immunoreactivity was again observed The NOS2 gene encodes inducible NOS (iNOS), one of three in the cell somas and apical dendrites of cortical and hippocam- NOS protein isoforms (iNOS, neuronal NOS, and endothelial NOS) that produce NO in the brain. Although commonly localized to macrophagic immune cells, iNOS is observed pri- Conflict of interest statement: No conflicts declared. marily in neurons and astrocytes in AD (10–12). Footprints of This paper was submitted directly (Track II) to the PNAS office. NO’s past presence include observation of nitrated in Freely available online through the PNAS open access option. AD brains, compared with normal age-matched brains (13), Abbreviations: AD, Alzheimer’s disease; APP, amyloid precursor protein; APPsw, APP suggesting that NO plays a role in the disease process. Our data Swedish mutant; A␤, ␤-amyloid; NOS, NO synthase; iNOS, inducible NOS. demonstrating hyperphophorylation of tau at disease-specific †C.A.C. and M.P.V. contributed equally to this work. sites, redistribution of tau to the somatodendritic compartment ‡To whom correspondence should be addressed. E-mail: [email protected]. of cortical and hippocampal neurons, and tau aggregates in © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601075103 PNAS ͉ August 22, 2006 ͉ vol. 103 ͉ no. 34 ͉ 12867–12872 Fig. 1. Loss of NOS2 RNA and activity. Brains from APPsw͞NOS2Ϫ/Ϫ mice were assayed for the expression of NOS2 mRNA (A) and calcium-independent NOS activity (B). Detection of GAPDH mRNA served as a loading control in A. *, P ϭ 0.05. Fig. 3. Western blot confirms the presence of hyperphosphorylated tau in ͞ Ϫ/Ϫ ϩ ͞ pal neurons. The neuronal pattern of immunostaining in APPsw NOS2 brains. (A) Total tau (Tau5 ) levels are similar in APPsw NOS2Ϫ/Ϫ brain compared with littermate NOS2Ϫ/Ϫ controls. (B) Western blot ͞ Ϫ/Ϫ APPsw NOS2 brains qualitatively resembled the AT180 im- for AT8 immunoreactive bands in APPsw͞NOS2Ϫ/Ϫ, TauϪ/Ϫ, P301L human tau munostaining pattern observed in JNPL3 mice with the P301L mutation, WT, and NOS2Ϫ/Ϫ mice. human tau mutation (Fig. 2I) that express hyperphosphorylated and aggregated tau (17). To determine whether tau protein levels were altered in the rylated tau. In contrast, each of three individual APPsw͞ APPsw͞NOS2Ϫ/Ϫ mice, we compared total tau expression by NOS2Ϫ/Ϫ brain samples demonstrated AT8 immunoreactivity. using Western blots and the Tau5 antibody that detects both Interestingly, low levels of AT8-positive tau were seen in brain phosphorylated and nonphosphorylated forms of tau. No dif- lysates from the NOS2Ϫ/Ϫ littermate controls. ference in total tau was observed between lysates from APPsw͞ To confirm the presence of aggregated tau, we used a filter NOS2Ϫ/Ϫ and NOS2Ϫ/Ϫ brains (Fig. 3A). Using the AT8 anti- assay (18) that traps protein aggregates present in brain lysates body, we examined brain lysates for the presence of on a cellulose filter. Tau5ϩ staining of trapped aggregates was hyperphosphorylated tau (Fig. 3B). Neither WT nor tau knock- observed with brain filtrates from NOS2Ϫ/Ϫ, APPsw͞NOS2Ϫ/Ϫ, out mice demonstrated bands corresponding to hyperphospho- and P301L mice, whereas no staining was observed with brain

Fig. 2. The APPsw͞NOS2Ϫ/Ϫ mouse demonstrates somatodendritic localization of hyperphosphorylated tau. (A–C and F) CA4 hippocampal (A) and globus palladius (B) neurons from an APPsw͞NOS2Ϫ/Ϫ mouse were immunopositive for tau phosphorylated at Ser-202͞Thr-205 by using the CP13 antibody or the AT8 antibody in neurons from frontal cortex (C) or hippocampus (F). Note the dense hyperphosphorylated tau immunoreactivity in soma and apical dendrites in B. (D and E) AT8 immunoreactivity was not observed in brain sections from littermate NOS2Ϫ/Ϫ mice (D; cortex) or APPsw mice (E; hippocampus). (G) Neurons from APPsw͞NOS2Ϫ/Ϫ brain were also immunopositive for tau phosphorylated at Thr-231 by using the AT180 antibody in the cortex. (H) No AT180 staining was observed in littermate NOS2Ϫ/Ϫ brains. (I) AT180 immunoreactivity in cortical sections from a mouse expressing the P301L human tau mutation was used as a positive control for hyperphosphorylated tau.

12868 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601075103 Colton et al. Fig. 5. Total and insoluble A␤ is increased in the APPsw͞NOS2Ϫ/Ϫ brain. (A) Amyloid deposits in brain sections from APPsw͞NOS2Ϫ/Ϫ mice were detected by using thioflavin S staining and immunoreactivity to the 4G8 antibody ͞ Ϫ/Ϫ Fig. 4. Aggregated tau proteins are observed in APPsw NOS2 brain. (A (Inset). (B–D)A␤ levels in brain lysates of the APPsw͞NOS2Ϫ/Ϫ mice were and B) Trapped aggregates from whole brain filtrates were immunoreactive compared with APPsw littermate controls by using an ELISA. Average values ͞ Ϫ/Ϫ Ϫ/Ϫ to Tau5 (A) and AT8 (B). APPsw Tau or Tau brains served as negative (Ϯ SEM) for the ratio of A␤40 to A␤42 (D), soluble and insoluble A␤ levels (C), controls, and P301L mouse brain served as positive control for the presence of and total A␤ levels (B) are shown. ns, no significant difference. tau aggregates. AT8 did not cross-react with A␤ aggregates formed by the addition of preaggregated A␤42 to brain lysates and then filtered. Filter- ␤ ␤ trapped A aggregates were detected by 4G8, an antibody against A peptide brains (Fig. 6). Head-injured APPsw mice served as a positive (data not shown). (C) Aggregates (Tau5ϩ) were also detected by using scan- ning EM. Tau5ϩ aggregates were observed in lysates from APPsw͞NOS2Ϫ/Ϫ, control and displayed numerous degenerating neurons. MEDICAL SCIENCES P301L mice, and autopsied AD brain samples. (D) Intracellular tau aggregates The mechanism of cell injury was further explored by evalu- were detected by using thioflavin S histochemistry. Fluorescent particles were ating markers for apoptosis. The activated form of caspase-3, a observed in neuronal somas from APPsw͞NOS2Ϫ/Ϫ brains, but not littermate known executioner caspase involved in apoptotic cell death (22), controls. was detected by using immunocytochemistry. Activated caspase-3 was observed in cell bodies and apical dendrites in cortical and hippocampal neurons in APPsw͞NOS2Ϫ/Ϫ brains filtrates from WT or tauϪ/Ϫ mice (Fig. 4A). AT8-immunoreac- (Fig. 7A). Slight, but observable, activated caspase-3 immuno- tive hyperphosphorylated tau was found in filter-trapped aggre- reactivity was found in NOS2Ϫ/Ϫ brains versus background gates from APPsw͞NOS2Ϫ/Ϫ and P301L brains, with only slight staining in APPsw or WT littermates. To detect whether acti- immunoreactivity observed from the NOS2Ϫ/Ϫ filtrate (Fig. 4B). vated caspase cleaved tau, we immunostained APPsw͞NOS2Ϫ/Ϫ Tau aggregation was further confirmed with scanning EM and brain sections with an antibody that recognizes tau truncated at thioflavin S histochemistry. Filters containing trapped tau ag- Asp-421 (TauC3) (23). TauC3-positive staining was observed in Ϫ Ϫ gregates were immunoreacted with Tau5 antibody, followed by cell bodies and dendrites of cortical neurons in APPsw͞NOS2 / an immunogold secondary antibody and silver enhancement to mice (Fig. 7E) compared with APPsw control brains (Fig. 7F). improve detection. Tau aggregates were clearly observed in brain Ϫ Ϫ Discussion filtrates from APPsw͞NOS2 / mice (Fig. 4C) and were com- parable to immunoreactive tau aggregates prepared from brains The amyloid and tau pathologies that characterize AD brain ͞ Ϫ/Ϫ of human AD or JNPL3 mice. Thioflavin S-positive aggregates lesions were simultaneously observed in the APPsw NOS2 were observed within the cell bodies of cortical neurons in the mouse brain. Unlike other common mouse models for amyloid APPsw͞NOS2Ϫ/Ϫ mice, but were not observed in NOS2Ϫ/Ϫ deposition, redistribution of normal mouse tau to the somato- dendritic region of cortical and hippocampal neurons, hyper- littermates (Fig. 4D). phosphorylation of mouse tau at multiple, disease-associated In addition to tau pathology, amyloid plaque-like pathology residues, and mouse tau aggregates were observed. These was observed in APPsw͞NOS2Ϫ/Ϫ brains. Amyloid deposits changes occurred in the presence of nonmutated mouse tau and could be detected by using 4G8, an antibody that reacts with are associated with neuronal degeneration in the cortex. Amy- human ␤-amyloid (A␤) peptides, or thioflavin S, a fluorescent ͞ ␤ loid plaque morphology and distribution in the APPsw indicator for -pleated sheet structures (Fig. 5A). To compare NOS2Ϫ/Ϫ brain was visually similar to that observed in APPsw ͞ Ϫ/Ϫ APPsw NOS2 with APPsw littermates, we directly measured littermates. However, we observed a significant increase in total ␤ ␤ soluble and insoluble A 40 and A 42 levels in brain lysates by brain A␤ peptides, which were primarily in the insoluble form. ␤ using a quantitative ELISA (19, 20). Total brain A levels were Increased levels of total A␤ peptides and altered ratios of A␤40 ͞ Ϫ/Ϫ Ϫ Ϫ significantly greater in APPsw NOS2 mice compared with to A␤42 in the APPsw͞NOS2 / brain suggest that NO acts on APPsw littermate controls (Fig. 5B). This increase was caused by A␤ generation or clearance, although the mechanism of inter- a significant increase in insoluble A␤ peptides, resulting in an action is unknown. increased A␤40͞A␤42 ratio (Fig. 5 C and D). Increased tau pathology and increased A␤ levels in our Although neuronal loss is not common in APPsw mice, we APPsw͞NOS2Ϫ/Ϫ mice were in contrast to the report of Nathan used Fluorojade B to identify degenerating neurons in the et al. (24). In that study, trigenic mice with the APP Swedish APPsw͞NOS2Ϫ/Ϫ brains (21). Widespread cortical neuronal double mutation (K670N, M671L), a mutated human presenilin damage was observed in three of four APPsw͞NOS2Ϫ/Ϫ mice gene (hPS1-A246E mutation), and a NOS2Ϫ/Ϫ background had compared with no apparent damage in either APPsw or WT an increased life span, decreased microglial activation, and an

Colton et al. PNAS ͉ August 22, 2006 ͉ vol. 103 ͉ no. 34 ͉ 12869 Fig. 7. Immunoreactivity for cleaved caspase-3 is increased in APPsw͞ NOS2Ϫ/Ϫ brain. (A–D) Cleaved caspase-3 immunoreactivity was increased in hippocampal neurons and their processes in the APPsw͞NOS2Ϫ/Ϫ brain (A) compared with NOS2Ϫ/Ϫ (B), APPsw (C), and WT (D) control mice. (C) NOS2Ϫ/Ϫ mice demonstrated low, but clearly observable, cleaved caspase immunore- activity, confirming published data demonstrating increased caspase-3 activ- ity in the NOS2Ϫ/Ϫ mouse brain (59, 60). (E and F) Neurons in the APPsw͞ NOS2Ϫ/Ϫ brain also demonstrated immunoreactivity for caspase-cleaved (truncated) tau by using the TauC3 antibody (E) compared with APPsw (F) brains.

Fig. 6. Degenerating neurons are observed in the APPsw͞NOS2Ϫ/Ϫ brain. Fluorojade B was used to detect degenerating neurons as described by and the subsequent phosphorylation of cGMP-dependent pro- Schmued et al. (21). (A) Degenerating neurons were observed in cortical tein kinases such as Ras-Raf-extracellular regulated kinase Ϫ Ϫ sections from APPsw͞NOS2 / mouse brains. (B and C) No degenerating and phosphatidylinositol 3-kinase-Akt (33, 34) play a critical neurons were observed with fluorojade B staining in brain sections from WT role in neuronal survival (35, 36). Neuronal survival is also (B) or APPsw (C) brains. (D) Intense staining was observed in a head-injured mediated by NO’s inhibition of executioner caspase-3 and APPsw control mouse brain. caspase-6 (37) where NO can block the conversion of the proenzymes via Akt-mediated phosphorylation or chemically age-dependent decrease in amyloid plaque burden, suggesting modify and decrease enzyme activity (38, 39). Higher levels of that NO is detrimental in these trigenics. However, the addition NO (Ͼ400 nM), as observed in eradication of pathogens of a mutated presenilin gene by Nathan et al. is a significant during an acute immune response, have pathophysiological difference from our APPsw͞NOS2Ϫ/Ϫ mice and may account for consequences. At pathogenic levels, p53 activation and the the opposing results. Hashimoto et al. (25) demonstrated that the induction of apoptosis can occur (33, 37). Taken together, the PS-1 A246E mutation promotes NO-mediated toxicity. As a temporal and spatial distribution of NO plays a vital role in consequence, removal of NO in PS-1 A246E mice would be controlling cell functions. predicted to decrease cell death and alter gamma secretase Our data suggest that maintenance of a critical level of NO at function to potentially reduce amyloid deposition. The subtype the neuron is essential to the survival programs that depend on of mutation thus may dictate differing requirements for NO. NO signaling pathways. The source of NO may not be as Furthermore, expression of mutated PS-1 in immune cells is important as the actual level of NO. In fact, changes in both NOS associated with a greatly enhanced inflammatory response that enzymes and NO scavengers were observed in AD. Fernandez- is not typical of sporadic AD (26). Because the immune response Vizarra et al. (40) have shown that neuronal NOS protein levels and the disease progression changes with age in AD (27), the aberrantly increase in AD neurons, whereas neuronal NOS brain’s requirement for NO may also fluctuate, enabling NO activity in brain lysates is reduced. Increased expression of function at specific disease stages to result in differing outcomes. endothelial NOS in AD is also observed, but is primarily Although NO is generally viewed as detrimental to neurons localized to the dystrophic neurites and astrocytes that surround (28), mounting evidence indicates a protective role for NO in amyloid plaques (11, 41). Our quantitative PCR data support the the brain (10, 29, 30). For example, Sinz et al. (31) have shown idea that changes in constitutive NOS isoforms are unlikely to that cognitive behavioral responses after head injury are worse adequately compensate for the genetic loss of iNOS. A fall in NO in iNOS knockout mice than in WT controls. Although these may be caused, in part, by a parallel increase in scavenging data support a beneficial function of iNOS in head trauma, mechanisms for NO. For example, redox active iron accumulates exogenous Abeta peptide-mediated synaptic damage in hip- in neurons in AD (42) and contributes to nitration of proteins pocampal slices is significantly reduced when NO is removed such as A␤ (43). The process of nitration, in turn, represents (32). Resolution of these disparate views about NO’s actions conditions that reduce NO bioavailability and alter cell signaling in the brain may reside in understanding specific concentration pathways initiated by specific levels of NO. and temporal profiles of NO release and the intracellular Interestingly, the changes in neuronal NOS and endothelial signaling pathways initiated by individual profiles. Low levels NOS levels and activities as AD progresses are associated with of NO (Ͻ100 nM) promote progrowth and antiapoptotic ectopic expression of iNOS within neurons (40). iNOS serves pathways (33). For example, activation of guanyl cyclase by NO as a widely diffusible and long-lasting source of NO that is most

12870 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601075103 Colton et al. commonly associated with activated immune cells such as Littermate controls were generated from the backcrossed microglia and astrocytes. Although contradictory data exist strain (Ϸ75% C57BL͞6 and 25% SJL͞129 SVJ). JNPL3 (TAU- (44), it has been generally believed that A␤ peptides induce P301L) mice were a generous gift from J. Lewis and M. Hutton iNOS that increases NO and kills surrounding neurons (45). (Mayo Clinic, Jacksonville, FL). All mice were genotyped by Microglia and astrocytes in AD brain, however, may not using standard procedures. produce those pathological levels of NO associated with LPS injection into the brain or with acute immune stimulation (29, Immunocytochemistry Antibodies. Hyperphosphorylated tau was 46, 47). Neuronal expression of iNOS may then serve to detected with the following antibodies: AT8-phospho-Ser-202͞ compensate for the integrated loss of NO in an attempt to Thr-205 (1:500; Pierce Biotechnology, Rockford, IL), CP13- retain NO’s protection in AD. phospo-Ser-202͞Thr-205 (1:600; a gift of Peter Davies, Albert Our data clearly indicate that genetic reduction in iNOS Einstein College of Medicine, Bronx, NY), and AT180- phos- ␤ activates caspase-3 in neurons in vivo. Although A peptide- phor-Thr-231 (1:500; Pierce Biotechnology). Total phosphory- mediated activation of caspase-3 and caspase-6 has been directly lated and nonphosphorylated tau was detected with Tau5 related to neuronal apoptosis in AD (48, 49), the lack of neuronal (1:3,000; Calbiochem, San Diego, CA), and A␤͞amyloid depos- ␤ death in mouse models that overexpress APP and A strongly its were detected with 4G8 (1:1,000; Senetek, Napa, CA). suggest that natural inhibitors of apoptosis must also be present. Activated caspase-3 was detected by using anti-activeR caspase-3 In addition to other apoptosis inhibitors found in neurons (49), (1:50; Cell Signaling Technology, Beverly, MA). Truncated tau NO may inhibit apoptosis. Caspase-3 activation may also directly was detected with TauC3 antibody [a generous gift of L. I. Binder impact tau pathology, because increased caspase-3 activation in (Northwestern University, Evanston, IL)]. the APPsw͞NOS2Ϫ/Ϫ brain is associated with tau truncation. This truncated form of tau appears to promote neurofibrillary Quantitative RT-PCR. Mouse brain RNA was extracted with the tangle formation (23, 48, 50), and loss of NO may enhance this Versagene RNA Purification system (Gentra Systems, Minne- truncation͞aggregation mechanism. apolis, MI) and converted to cDNA by using a High-Capacity NO’s biological activity is diverse, and alternative mechanisms of cDNA archive kit (Applied Biosystems, Foster City, CA). NOS2 tangle formation may also exist. Reynolds et al. (51) reported that ࿝ specific N-terminal tyrosine residues of tau are nitrated, that these mRNA (GenBank accession no. NM 010927) expression was identified with primer a (5Ј-GCATCCCAAGTACGAGTGGT- nitrotyrosines inhibit tau aggregation, and thus may protect against Ј ͞ pathological tau deposition (50). By this mechanism, the decreased 3 , spanning the exon9 exon 10 boundary to ensure no ampli-

Ј MEDICAL SCIENCES NO in APPsw͞NOS2Ϫ/Ϫ mice would reduce tau nitration and fication of genomic DNA) and primer b (5 -ATTCTGCCA- Ј promote tau aggregation (52, 53). Alternatively, the activity of GATGTGGGTCTTCCA-3 ). glycogen synthase kinase 3␤ (GSK-3␤) that phosphorylates tau is regulated by Akt-mediated signaling, which is regulated by NO, NOS Activity. NOS enzyme activity was measured by the conver- potentially via soluble guanylate cyclase activation and cGMP. NO sion of L-arginine to L-citrulline as described (58) and expressed increases the activity of Akt, which in turn inhibits the phosphor- as pmol of L-14C-citrulline produced per mg of protein. ylation of GSK-3␤. When NO is reduced, the inhibitory pathway over kinase function is also reduced. As a principal kinase involved Aggregate Filter Assay. Aggregate levels were measured with a in tau hyperphosphorylation (8, 54), loss of this regulatory effect of filter retardation assay (18) and immunodetection for either NO on GSK-3␤ activity may promote tau pathology. Other kinases total tau (Tau5) or hyperphosphorylated tau (AT8). involved in tau phosphorylation may also be affected by NO- mediated events. Scanning EM. Brain aggregates retained on filters as described were In summary, APPsw͞NOS2Ϫ/Ϫ mice provide clear genetic immunostained with Tau5 antibody and detected with goat anti- data that removal of a major synthetic source of NO over a mouse IgG conjugated with 40 nM gold particles (1:20; Ted Pella, lifetime of exposure to A␤ peptides promotes tau pathology in Redding, CA) and a silver enhancing kit (Ted Pella). A Phillips KL the brain. Our data also suggest that A␤, in the presence of 30 environmental scanning electron microscope at the Duke Bio- reduced NO, may be instrumental in the production of hyper- logical Science Environmental Scanning Electron Microscope fa- phosphorylated and aggregated tau, thereby regulating the cility was used for imaging with the kind assistance of Leslie Eibest. merger of the two pathologies into the amyloid cascade hypoth- esis of Selkoe and coworkers (55, 56). The potential for NO to Detection of Soluble and Insoluble A␤. Soluble and insoluble pools act as an inhibitory modulator of caspase activity places NO at of A␤40 and A␤42 were measured with a specific ELISA and a junction point between A␤ peptides, caspase cleavage of tau, differential brain extractions as described (19, 20). and tau aggregation. At the least, the APPsw͞NOS2Ϫ/Ϫ mouse provides a tool to further our understanding of the role of Statistics. Average values Ϯ SEM were calculated for quanti- NO-mediated events in AD. tative PCR and ELISA data (n ϭ 3–7 animals per group). Statistical significance was calculated by using the unpaired Methods Student’s t test with the Prism 3.02 program (GraphPad, San Mouse Strains. A bigenic mouse was produced by crossing Tg Diego, CA). (HuAPP695.K670N-M671L)2576 mice with NOS2Ϫ/Ϫ (B6 129P2NOS2tau1Lau͞J) (Jackson Laboratory, Bar Harbor, ME) Ϫ Ϫ We thank Dr. C. M. Hulette and J. Ervin of the Kathleen Bryan Brain mice. Phenotypes of APPsw and NOS2 / mice have been Bank at Duke University for AD and normal control brain tissues. This described (1, 57). Tg2576 mice were a generous gift of K. work was supported by National Institutes of Health Grants AG19780 (to Hsaio-Ashe (University of Minnesota, Minneapolis, MN). M.P.V.) and AG19740 (to C.A.C.).

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12872 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601075103 Colton et al.