An increase in L-type voltage-gated calcium channel (LTCC) current is a prominent biomarker of brain aging and is believed to contribute to cognitive decline and vulnerability to neuropathologies. Studies examining age-related changes in LTCCs have focused primarily on males, although estrogen (17β-estradiol, E2) affects calcium-dependent activities associated with cognition. Therefore, to better understand brain aging in females, the effects of chronic E2 replacement on LTCC current activity in hippocampal neurons of young and aged ovariectomized rats were determined. The zipper slice preparation was used to expose cornu ammonis 1 (CA1) pyramidal neurons for recording LTCC currents using the cell-attached patch-clamp technique. We found that an age-related increase in LTCC current in neurons from control animals was prevented by E2 treatment. In addition, in situ hybridization revealed that within stratum pyramidale of the CA1 area, mRNA expression of the Ca v 1.2 LTCC subunit, but not the Ca v 1.3 subunit, was decreased in aged E2-treated rats. Thus, the reported benefits of E2 on cognition and neuronal health may be attributed, at least in part, to its age-related decrease in LTCC current.
Thyroid hormone is essential for mammalian brain development, but the mechanisms by which thyroid hormone exerts its effects, the developmental processes affected, and the timing of thyroid hormone effects are poorly understood. An important question is whether thyroid hormone of maternal origin is essential in guiding fetal brain development. In both humans and rats, thyroid hormone of maternal origin reaches the fetus before the onset of fetal thyroid function. Moreover, receptors for thyroid hormone (TRs) are present in the fetal brain and are occupied by thyroid hormone. Finally, a recent report strongly indicates that transient undiagnosed maternal hypothyroidism can lead to measurable neurological deficits in the offspring despite the lack of neonatal hypothyroidism. Considering that TRs are ligand-activated transcription factors, we recently initiated a project to identify thyroid hormone-responsive genes in the fetal cortex before the onset of fetal thyroid function. One of the thyroid hormone-responsive genes we identified, neuroendocrine-specific protein (NSP), is expressed as two separate transcripts, NSP-A and NSP-C. Only NSP-A is affected by maternal thyroid hormone. We now demonstrate that the messenger RNA encoding NSP-A is expressed exclusively in the proliferative zone of the fetal cortex, and that its expression is affected by maternal hypothyroidism. Moreover, as development proceeds, NSP-A becomes selectively expressed in Purkinje cells of the cerebellum, a well known thyroid hormone-responsive cell. These findings strongly support the concept that thyroid hormone of maternal origin exerts specific receptor-mediated effects on fetal brain development.
Human studies suggest either a protective role or no benefit of statins against the development of Alzheimer's disease (AD). We tested the hypothesis that statin-mediated cholesterol reduction in aged dogs, which have cognitive impairments and amyloid-β (Aβ) pathology, would improve cognition and reduce neuropathology. In a study of 12 animals, we treated dogs with 80 mg/day of atorvastatin for 14.5 months. We did not observe improvements in discrimination learning; however, there were transient impairments in reversal learning, suggesting frontal dysfunction. Spatial memory function did not change with treatment. Peripheral levels of cholesterol, LDLs, triglycerides, and HDL were significantly reduced in treated dogs. Aβ in cerebrospinal fluid and brain remained unaffected. However, β-secretase-1 (BACE1) protein levels and activity decreased and correlated with reduced brain cholesterol. Finally, lipidomic analysis revealed a significant decrease in the ratio of omega-6 to omega-3 essential fatty in temporal cortex of treated aged dogs. Aged beagles are a unique model that may provide novel insights and translational data that can predict outcomes of statin use in human clinical trials. Treatment with atorvastatin may be beneficial for brain aging by reducing BACE1 protein and omega6:omega3 ratio, however, the potential adverse cognitive outcomes reported here should be more carefully explored given their relevance to human clinical outcomes.
We recently demonstrated that a variant allele of CYP3A5 (CYP3A5*3) confers low CYP3A5 expression as a result of improper mRNA splicing. In this study, we further evaluated the regulation of CYP3A5 in liver and jejunal mucosa from white donors. For all tissues, high levels of CYP3A5 protein were strongly concordant with the presence of a wild-type allele of the CYP3A5 gene (CYP3A5*1). CYP3A5 represented greater than 50% of total CYP3A content in nearly all of the livers and jejuna that carried the CYP3A5*1 wild-type allele. Overall, CYP3A5 protein content accounted for 31% of the variability in hepatic midazolam hydroxylation activity. Improperly spliced mRNA (SV1-CYP3A5) was found only in tissues containing a CYP3A5*3 allele. Properly spliced CYP3A5 mRNA (wt-CYP3A5) was detected in all tissues, but the median wt-CYP3A5 mRNA was 4-fold higher in CYP3A5*1/*3 livers compared with CYP3A5*3/*3 livers. Differences in wt-CYP3A5 and CYP3A4 mRNA content explained 53 and 51% of the interliver variability in CYP3A5 and CYP3A4 content, respectively. Hepatic CYP3A4 and CYP3A5 contents were not correlated when all livers were compared. However, for CYP3A5*1/*3 livers, levels of the two proteins were strongly correlated (r = 0.93) as were wt-CYP3A5 and CYP3A4 mRNA (r = 0.76). These findings suggest that CYP3A4 and CYP3A5 genes share a common regulatory pathway for constitutive expression, possibly involving conserved elements in the 5'-flanking region.
β-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-β (Aβ) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: β-site Aβ precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aβ deposition. Thus, expression of both forms of β-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics. β-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-β (Aβ) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: β-site Aβ precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aβ deposition. Thus, expression of both forms of β-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics. Alzheimer's disease (AD) is the most common age-related neurodegenerative disease. Although progressive memory loss is the best known clinical symptom, patients also exhibit a wide range of other behavioral disturbances, from paranoia and delusions to a gradual decline in language ability. In the affected brain, two forms of neuropathology develop in characteristic patterns: extracellular amyloid deposits, referred to as plaques, and intracellular neurofibrillary tangles (NFTs). The presence of all three—NFTs, plaques, and cognitive impairment—define AD as a clinical entity. The lack of an effective, disease-modifying therapy for AD is one of the greatest unmet medical needs facing modern society. Despite gaps in our knowledge about the general causes of AD, much is known about the molecular biology underlying several key pathways. A large body of compelling evidence provides broad support for the idea that the amyloid-β (Aβ) peptide is the causative pathologic agent in AD. The Aβ peptide is produced from the larger Aβ precursor protein (APP) through two sequential enzymatic activities, β- and γ-secretase. β-Secretase first cleaves APP to release a large secreted derivative, sAPPβ. A membrane-bound fragment, C-terminal fragment of APP (CTF) β, remains and is, in turn, proteolytically cleaved by γ-secretase to generate Aβ and a cytosolic fragment, which may offer a clue to the physiologic function of APP.1Cao X. Sudhof T.C. A transcriptively active complex of APP with Fe65 and histone acetyltransferase Tip60.Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar β-Secretase is the rate-limiting enzyme in the production of Aβ, a limitation that likely arises because it does not often encounter its APP substrate in the cell.2Capell A. Meyn L. Fluhrer R. Teplow D.B. Walter J. Haass C. Apical sorting of β-secretase limits amyloid β-peptide production.J Biol Chem. 2002; 277: 5637-5643Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Consequently, targeting β-secretase is considered a prime candidate strategy for developing an effective AD therapy. The atypical aspartyl protease β-site APP cleaving enzyme (BACE1) was identified as the major β-secretase enzyme by multiple research groups slightly more than a decade ago.3Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. β-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.Science. 1999; 286: 735-741Crossref PubMed Scopus (3329) Google Scholar Mature BACE1 is a palmitoylated glycoprotein that is processed to an active form by removal of a prodomain by a furin-like convertase activity.4Capell A. Steiner H. Willem M. Kaiser H. Meyer C. Walter J. Lammich S. Multhaup G. Haass C. Maturation and pro-peptide cleavage of β-secretase.J Biol Chem. 2000; 275: 30849-30854Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar BACE1 is involved in nervous system myelination,5Willem M. Garratt A.N. Novak B. Citron M. Kaufmann S. Rittger A. DeStrooper B. Saftig P. Birchmeier C. Haass C. Control of peripheral nerve myelination by the β-secretase BACE1.Science. 2006; 314: 664-666Crossref PubMed Scopus (614) Google Scholar and increases in BACE1 after traumatic brain injury may reflect this function.6Loane D.J. Pocivavsek A. Moussa C.E. Thompson R. Matsuoka Y. Faden A.I. Rebeck G.W. Burns M.P. Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury.Nat Med. 2009; 15: 377-379Crossref PubMed Scopus (200) Google Scholar The BACE1 cleavage product of APP (sAPPβ), may have distinct physiologic functions that distinguish it from the α-secretase product (sAPPα).7Li H. Wang B. Wang Z. Guo Q. Tabuchi K. Hammer R.E. Sudhof T.C. Zheng H. Soluble amyloid precursor protein (APP) regulates transthyretin and Klotho gene expression without rescuing the essential function of APP.Proc Natl Acad Sci U S A. 2010; 107: 17362-17367Crossref PubMed Scopus (98) Google Scholar There are five BACE1 splice variants, although it is unknown whether their variation is related to the development of AD.8Zohar O. Cavallaro S. D'Agata V. Alkon D.L. Quantification and distribution of β-secretase alternative splice variants in the rat and human brain.Brain Res Mol Brain Res. 2003; 115: 63-68Crossref PubMed Scopus (43) Google Scholar Considerably less is known about the homologous enzyme BACE2. BACE1 and BACE2 compete for substrate and can both cleave APP at the β-site.9Hussain I. Powell D.J. Howlett D.R. Chapman G.A. Gilmour L. Murdock P.R. Tew D.G. Meek T.D. Chapman C. Schneider K. Ratcliffe S.J. Tattersall D. Testa T.T. Southan C. Ryan D.M. Simmons D.L. Walsh F.S. Dingwall C. Christie G. ASP1 (BACE2) cleaves the amyloid precursor protein at the β-secretase site.Mol Cell Neurosci. 2000; 16: 609-619Crossref PubMed Scopus (144) Google Scholar, 10Basi G. Frigon N. Barbour R. Doan T. Gordon G. McConlogue L. Sinha S. Zeller M. Antagonistic effects of β-site amyloid precursor protein-cleaving enzymes 1 and 2 on β-amyloid peptide production in cells.J Biol Chem. 2003; 278: 31512-31520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Although BACE2 is present in most peripheral tissues,11Bennett B.D. Babu-Khan S. Loeloff R. Louis J.C. Curran E. Citron M. Vassar R. Expression analysis of BACE2 in brain and peripheral tissues.J Biol Chem. 2000; 275: 20647-20651Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar there is a moderate amount of BACE2 expression in the human brain.12Maloney B. Ge Y.W. Greig N.H. Lahiri D.K. Characterization of the human β-secretase 2 (BACE2) 5′-flanking region: identification of a 268-bp region as the basal BACE2 promoter.J Mol Neurosci. 2006; 29: 81-99Crossref PubMed Scopus (17) Google Scholar In the brain, BACE2 is believed to be mostly astrocytic, whereas BACE1 is largely neuronal.10Basi G. Frigon N. Barbour R. Doan T. Gordon G. McConlogue L. Sinha S. Zeller M. Antagonistic effects of β-site amyloid precursor protein-cleaving enzymes 1 and 2 on β-amyloid peptide production in cells.J Biol Chem. 2003; 278: 31512-31520Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar BACE2 is located on chromosome 21 in the Down's syndrome (DS) obligate region and may contribute to amyloid abnormalities in these individuals.13Acquati F. Accarino M. Nucci C. Fumagalli P. Jovine L. Ottolenghi S. Taramelli R. The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region.FEBS Lett. 2000; 468: 59-64Crossref PubMed Scopus (121) Google Scholar, 14Motonaga K. Itoh M. Becker L.E. Goto Y. Takashima S. Elevated expression of β-site amyloid precursor protein cleaving enzyme 2 in brains of patients with Down syndrome.Neurosci Lett. 2002; 326: 64-66Crossref PubMed Scopus (35) Google Scholar Knockout of BACE1 in the mouse leads to the abolishment of Aβ, sAPPβ, and CTFβ production in the brain.15Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.-C. Yan Q. Richards W.G. Citron M. Vassar R. Mice deficient in BACE1, the Alzheimer's β-secretase, have normal phenotype and abolished β-amyloid generation.Nature neuroscience. 2001; 4: 231-232Crossref PubMed Scopus (956) Google Scholar Crossing BACE1 knockout mice with models of amyloid deposition leads to reduced brain Aβ and improved cognitive function.16Ohno M. Cole S.L. Yasvoina M. Zhao J. Citron M. Berry R. Disterhoft J.F. Vassar R. BACE1 gene deletion prevents neuron loss and memory deficits in 5XFAD APP/PS1 transgenic mice.Neurobiol Dis. 2007; 26: 134-145Crossref PubMed Scopus (247) Google Scholar However, BACE1 knockout animals have many subtle phenotypic alterations, including deficits in long-term potentiation, increased seizure susceptibility, and schizophrenic behavior.17Hu X. Zhou X. He W. Yang J. Xiong W. Wong P. Wilson C.G. Yan R. BACE1 deficiency causes altered neuronal activity and neurodegeneration.J Neurosci. 2010; 30: 8819-8829Crossref PubMed Scopus (128) Google Scholar, 18Savonenko A.V. Melnikova T. Laird F.M. Stewart K.A. Price D.L. Wong P.C. Alteration of BACE1-dependent NRG1/ErbB4 signaling and schizophrenia-like phenotypes in BACE1-null mice.Proc Natl Acad Sci U S A. 2008; 105: 5585-5590Crossref PubMed Scopus (249) Google Scholar BACE1 knockouts exhibit residual β-secretase activity that may be attributable to BACE2.15Luo Y. Bolon B. Kahn S. Bennett B.D. Babu-Khan S. Denis P. Fan W. Kha H. Zhang J. Gong Y. Martin L. Louis J.-C. Yan Q. Richards W.G. Citron M. Vassar R. Mice deficient in BACE1, the Alzheimer's β-secretase, have normal phenotype and abolished β-amyloid generation.Nature neuroscience. 2001; 4: 231-232Crossref PubMed Scopus (956) Google Scholar, 19Cai H. Wang Y. McCarthy D. Wen H. Borchelt D.R. Price D.L. Wong P.C. BACE1 is the major β-secretase for generation of Aβ peptides by neurons.Nature neuroscience. 2001; 4: 233-234Crossref PubMed Scopus (958) Google Scholar, 20Dominguez D. Tournoy J. Hartmann D. Huth T. Cryns K. Deforce S. Serneels L. Camacho I.E. Marjaux E. Craessaerts K. Roebroek A.J. Schwake M. D'Hooge R. Bach P. Kalinke U. Moechars D. Alzheimer C. Reiss K. Saftig P. De Strooper B. Phenotypic and biochemical analyses of BACE1- and BACE2-deficient mice.J Biol Chem. 2005; 280: 30797-30806Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar Our understanding of the relationship between BACE1 and BACE2, and how they may be connected in human disease, is incomplete. The amount of active BACE1 protein is elevated in the brain in late-stage AD,21Li R. Lindholm K. Yang L.B. Yue X. Citron M. Yan R. Beach T. Sue L. Sabbagh M. Cai H. Wong P. Price D. Shen Y. Amyloid β peptide load is correlated with increased β-secretase activity in sporadic Alzheimer's disease patients.Proc Natl Acad Sci U S A. 2004; 101: 3632-3637Crossref PubMed Scopus (447) Google Scholar, 22Ahmed R.R. Holler C.J. Webb R.L. Li F. Beckett T.L. Murphy M.P. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.J Neurochem. 2010; 112: 1045-1053Crossref PubMed Scopus (85) Google Scholar, 23Fukumoto H. Cheung B.S. Hyman B.T. Irizarry M.C. β-secretase protein and activity are increased in the neocortex in Alzheimer disease.Arch Neurol. 2002; 59: 1381-1389Crossref PubMed Scopus (600) Google Scholar, 24Holsinger R.M. McLean C.A. Beyreuther K. Masters C.L. Evin G. Increased expression of the amyloid precursor β-secretase in Alzheimer's disease.Ann Neurol. 2002; 51: 783-786Crossref PubMed Scopus (496) Google Scholar, 25Yang L.B. Lindholm K. Yan R. Citron M. Xia W. Yang X.L. Beach T. Sue L. Wong P. Price D. Li R. Shen Y. Elevated β-secretase expression and enzymatic activity detected in sporadic Alzheimer disease.Nat Med. 2003; 9: 3-4Crossref PubMed Scopus (601) Google Scholar and higher levels of BACE1 are also observed in the cerebrospinal fluid.26Holsinger R.M. Lee J.S. Boyd A. Masters C.L. Collins S.J. CSF BACE1 activity is increased in CJD and Alzheimer disease versus [corrected] other dementias.Neurology. 2006; 67: 710-712Crossref PubMed Scopus (69) Google Scholar Individuals with mild cognitive impairment (MCI), presumed to be the earliest detectable clinical phase of AD, also show elevated BACE1 expression in cerebrospinal fluid27Zetterberg H. Andreasson U. Hansson O. Wu G. Sankaranarayanan S. Andersson M.E. Buchhave P. Londos E. Umek R.M. Minthon L. Simon A.J. Blennow K. Elevated cerebrospinal fluid BACE1 activity in incipient Alzheimer disease.Arch Neurol. 2008; 65: 1102-1107Crossref PubMed Scopus (200) Google Scholar and increased β-secretase activity from platelets.28Liu W.W. Todd S. Craig D. Passmore A.P. Coulson D.T. Murphy S. Irvine G.B. Johnston J.A. Elevated platelet β-secretase activity in mild cognitive impairment.Dement Geriatr Cogn Disord. 2007; 24: 464-468Crossref PubMed Scopus (20) Google Scholar These studies have raised the intriguing possibility that changes in β-secretase activity may be directly connected to the development of AD; however, important questions remain unanswered. To be causally involved in AD, the increase in BACE1 should occur in the brain at an early, preclinical stage. This has not been conclusively established. Furthermore, minimal information is available on how specific the changes in BACE1 are to AD compared with other neurologic diseases.26Holsinger R.M. Lee J.S. Boyd A. Masters C.L. Collins S.J. CSF BACE1 activity is increased in CJD and Alzheimer disease versus [corrected] other dementias.Neurology. 2006; 67: 710-712Crossref PubMed Scopus (69) Google Scholar Finally, the function of BACE2 in the brain, and its possible role in disease, is unresolved. We recently observed unexpectedly high levels of active BACE2 protein in the AD brain,22Ahmed R.R. Holler C.J. Webb R.L. Li F. Beckett T.L. Murphy M.P. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.J Neurochem. 2010; 112: 1045-1053Crossref PubMed Scopus (85) Google Scholar raising the question of whether it may have been overlooked in earlier studies. In this study, we attempted to answer these questions by conducting a detailed examination of BACE1 and BACE2 in a broad cohort of patients. We selected two series of cases to study two overlapping questions (Table 1). The first case series (see Supplemental Table S1 at http://ajp.amjpathol.org) was chosen to examine the role of β-secretase in age-related neurodegenerative disease. These samples were obtained from the tissue repository of the Alzheimer's Disease Center at the University of Kentucky, Sanders-Brown Center on Aging. Controls (n = 9) were age matched to disease-affected cases [preclinical AD (PCAD): n = 10; amnestic MCI: n = 7; and AD: n = 10]. PCAD cases (often referred to as high-pathology controls) were defined as those that met the National Institute on Aging–Reagan Institute neuropathology criteria for likely AD but exhibited no clinical signs of dementia.29Price J.L. McKeel Jr, D.W. Buckles V.D. Roe C.M. Xiong C. Grundman M. Hansen L.A. Petersen R.C. Parisi J.E. Dickson D.W. Smith C.D. Davis D.G. Schmitt F.A. Markesbery W.R. Kaye J. Kurlan R. Hulette C. Kurland B.F. Higdon R. Kukull W. Morris J.C. Neuropathology of nondemented aging: presumptive evidence for preclinical Alzheimer disease.Neurobiol Aging. 2009; 30: 1026-1036Crossref PubMed Scopus (498) Google Scholar Amnestic MCI was defined as per the criteria of Petersen et al.30Petersen R.C. Smith G.E. Waring S.C. Ivnik R.J. Tangalos E.G. Kokmen E. Mild cognitive impairment: clinical characterization and outcome.Arch Neurol. 1999; 56: 303-308Crossref PubMed Scopus (7277) Google Scholar We included six cases of frontotemporal dementia (FTD) as an additional neurodegenerative disease and specificity control.31Cairns N.J. Bigio E.H. Mackenzie I.R. Neumann M. Lee V.M. Hatanpaa K.J. White CL III, C.L. Schneider J.A. Grinberg L.T. Halliday G. Duyckaerts C. Lowe J.S. Holm I.E. Tolnay M. Okamoto K. Yokoo H. Murayama S. Woulfe J. Munoz D.G. Dickson D.W. Ince P.G. Trojanowski J.Q. Mann D.M. Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration.Acta Neuropathol. 2007; 114: 5-22Crossref PubMed Scopus (908) Google Scholar Aβ is not considered to play a significant role in FTD. Details of the recruitment, inclusion criteria, and mental status test battery for the control group have been described previously.32Schmitt F.A. Davis D.G. Wekstein D.R. Smith C.D. Ashford J.W. Markesbery W.R. "Preclinical" AD revisited: neuropathology of cognitively normal older adults.Neurology. 2000; 55: 370-376Crossref PubMed Scopus (322) Google Scholar Details of the tissue collection procedures and consensus diagnosis have also been described (see Supplemental Figure S1 at http://ajp.amjpathol.org).33Nelson P.T. Abner E.L. Schmitt F.A. Kryscio R.J. Jicha G.A. Smith C.D. Davis D.G. Poduska J.W. Patel E. Mendiondo M.S. Markesbery W.R. Modeling the association between 43 different clinical and pathological variables and the severity of cognitive impairment in a large autopsy cohort of elderly persons.Brain Pathol. 2010; 20: 66-79Crossref PubMed Scopus (165) Google Scholar The second case series (see Supplemental Table S2 at http://ajp.amjpathol.org) was selected to better elucidate the role of Aβ deposition as a feedback mechanism for changes in β-secretase. Individuals with DS develop Aβ deposition and other AD-like abnormalities with age. DS cases and controls were obtained from the Alzheimer's Disease Center of the University of California at Irvine brain tissue repository and the National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders (University of Maryland). Control brains had no history of antemortem dementia. An analysis of RNA integrity numbers, controlling for age and postmortem interval, indicated no significant difference in tissue quality between sites (n = 46; F1,42 = 0.9, P < 0.4).Table 1Subject Demographic DataSex, M/F (no.)Age (years)⁎Values are given as mean ± SD.PMI (hours)⁎Values are given as mean ± SD.Brain weight (g)⁎Values are given as mean ± SD.Braak stage (median)MMSE score⁎Values are given as mean ± SD.Case series I Control5/484.3 ± 5.12.8 ± 0.81234 ± 163I28.4 ± 1.5 FTD3/361.0 ± 14.64.8 ± 1.0941 ± 16907.8 ± 9.0 Amnestic MCI3/489.0 ± 5.82.8 ± 0.51118 ± 132IV24.8 ± 3.1 Preclinical AD1/985.6 ± 3.72.6 ± 0.51173 ± 110IV29.4 ± 0.7 AD4/683.4 ± 5.73.1 ± 0.61074 ± 89VI9.9 ± 6.0Case series II Control10/1037.0 ± 21.89.5 ± 5.5NANANA DS18/1034.4 ± 21.213.5 ± 8.5NANANASeries I measurements were made from the SMTG (areas 21 and 22) and the CB; series II measurements were made from the middle frontal gyrus (area 9).F, female; M, male; MMSE, Mini-Mental State Examination; NA, not applicable or unavailable; PMI, postmortem interval. Values are given as mean ± SD. Open table in a new tab Series I measurements were made from the SMTG (areas 21 and 22) and the CB; series II measurements were made from the middle frontal gyrus (area 9). F, female; M, male; MMSE, Mini-Mental State Examination; NA, not applicable or unavailable; PMI, postmortem interval. Frozen samples were homogenized using a PowerMax Advanced Homogenizing System 200 (VWR, Batavia, IL) in five volumes (wet w/v) of tissue lysis buffer [10 mmol/L sodium acetate (NaOAc), 3.0 mmol/L NaCl, 0.1% Triton X-100 (Roche Diagnostics GmbH, Mannheim, Germany), 0.32 mol/L sucrose, pH 5.0]. The buffer was supplemented with a complete protease inhibitor cocktail with EDTA (PIC; Amresco, Solon, OH), with 100 nmol/L pepstatin A added (Sigma-Aldrich; St. Louis, MO). Whole tissue homogenate was centrifuged at ∼2000 × g for 15 minutes to pellet insoluble material, followed by an additional spin at 20,000 × g for 30 minutes. Pelleted material was sequentially extracted in an equal volume of radioimmunoprecipitation assay (RIPA) buffer (0.1% SDS, 0.5% deoxycholate, 1.0% Triton X-100, 50 mmol/L Tris base, 150 mmol/L NaCl, pH 8.0, with PIC) or 2% SDS (w/v, with PIC) to determine detergent-soluble Aβ, followed by 70% (v/v) formic acid (FA) to determine insoluble Aβ.34Beckett T.L. Niedowicz D.M. Studzinski C.M. Weidner A.M. Webb R.L. Holler C.J. Ahmed R.R. LeVine 3rd, H. Murphy M.P. Effects of nonsteroidal anti-inflammatory drugs on amyloid-β pathology in mouse skeletal muscle.Neurobiol Dis. 2010; 39: 449-456Crossref PubMed Scopus (20) Google Scholar In each case, the pellet was extracted by brief sonication (10 × 0.5-second microtip pulses at 20% power; Fisher sonic dismembrator, model 500, Fisher Scientific, Pittsburgh, PA) followed by centrifugation to pellet-insoluble material (detergent-soluble fraction: 20,000 × g for 30 minutes at 14°C; FA fraction: 20,000 × g for 1 hour at 4°C). Protein content was determined by bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL). For immunofluorescence, sections were labeled for BACE1 (rabbit monoclonal EPR3956; Epitomics Inc., Burlingame, CA), BACE2 (rabbit polyclonal Ab1; EMD Biosciences, San Diego, CA), glial fibrillary acidic protein (for astrocytes; clone GA5, MAB360; Millipore, Billerica, MA), Iba-1 (for microglia; Biocare Medical, Concord, CA), 4G8 (for amyloid deposits; Covance, Denver, PA), or PHF-1 (for pathologic tau; a gift from Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY) (see Supplemental Table S3 at http://ajp.amjpathol.org for a detailed list of all the antibodies used in this study). Sections were deparaffinized in SafeClear (Fisher Scientific) and rehydrated before blocking endogenous peroxidase activity with 3% hydrogen peroxide in 10% methanol. All the sections were subjected to antigen retrieval by boiling for 10 minutes in citric acid buffer (10 mmol/L sodium citrate + 0.05% Tween 20, pH 5.0) and blocked (PBS + 2.5% normal goat serum, 2.5% normal donkey serum, and 0.05% Tween 20). Secondary antibodies were anti-rabbit Alexa Fluor 568 (red) or anti-mouse Alexa Fluor 488 (green) (Invitrogen, Carlsbad, CA). Slides were treated with autofluorescence eliminator reagent (Millipore) and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA). Alternatively, we used the Vector avidin-biotin complex kit (Vector Laboratories) with either 3,3′-diaminobenzidine or Vector SG (Vector Laboratories) as substrate. We recently described and validated the assays for BACE1 and BACE2 for human tissue in considerable detail, including reagent specificity.22Ahmed R.R. Holler C.J. Webb R.L. Li F. Beckett T.L. Murphy M.P. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.J Neurochem. 2010; 112: 1045-1053Crossref PubMed Scopus (85) Google Scholar This method is similar to that of Fukumoto et al.35Fukumoto H. Rosene D.L. Moss M.B. Raju S. Hyman B.T. Irizarry M.C. β-Secretase activity increases with aging in human, monkey, and mouse brain.Am J Pathol. 2004; 164: 719-725Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar Determination of BACE1 (MAB931, raised against the BACE1 ectodomain; R&D Systems, Minneapolis, MN) or BACE2 (Ab1, raised against amino acids 496–511 of BACE2; EMD Biosciences) activities was as described.22Ahmed R.R. Holler C.J. Webb R.L. Li F. Beckett T.L. Murphy M.P. BACE1 and BACE2 enzymatic activities in Alzheimer's disease.J Neurochem. 2010; 112: 1045-1053Crossref PubMed Scopus (85) Google Scholar In some cases, validation experiments were performed using two different antibodies directed against the opposite ends of BACE1 (C-terminus, EPR3956; Epitomics Inc.) or BACE2 (N-terminus, rabbit polyclonal Ab2; EMD Biosciences). For neprilysin (NEP) activity, tissue samples were homogenized in ∼10 volumes of 50 mmol/L potassium phosphate buffer, pH 7.3. Samples were centrifuged at 500 × g for 15 minutes at 4°C, and the supernatant was used to measure NEP activity.36Shinall H. Song E.S. Hersh L.B. Susceptibility of amyloid β peptide degrading enzymes to oxidative damage: a potential Alzheimer's disease spiral.Biochemistry. 2005; 44: 15345-15350Crossref PubMed Scopus (95) Google Scholar Specificity was demonstrated by inhibition with 100 μmol/L phosphoramidon or 2 μmol/L CGS24592.37De Lombaert S. Erion M.D. Tan J. Blanchard L. el-Chehabi L. Ghai R.D. Sakane Y. Berry C. Trapani A.J. N-Phosphonomethyl dipeptides and their phosphonate prodrugs, a new generation of neutral endopeptidase (NEP, EC 3.4.24.11) inhibitors.J Med Chem. 1994; 37: 498-511Crossref PubMed Scopus (80) Google Scholar Detergent- and FA-soluble pools of Aβ were measured in tissue samples using a standard, well-characterized sandwich enzyme-linked immunosorbent assay (ELISA).34Beckett T.L. Niedowicz D.M. Studzinski C.M. Weidner A.M. Webb R.L. Holler C.J. Ahmed R.R. LeVine 3rd, H. Murphy M.P. Effects of nonsteroidal anti-inflammatory drugs on amyloid-β pathology in mouse skeletal muscle.Neurobiol Dis. 2010; 39: 449-456Crossref PubMed Scopus (20) Google Scholar, 38McGowan E. Pickford F. Kim J. Onstead L. Eriksen J. Yu C. Skipper L. Murphy M.P. Beard J. Das P. Jansen K. Delucia M. Lin W.L. Dolios G. Wang R. Eckman C.B. Dickson D.W. Hutton M. Hardy J. Golde T. Aβ42 is essential for parenchymal and vascular amyloid deposition in mice.Neuron. 2005; 47: 191-199Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 39Head E. Pop V. Sarsoza F. Kayed R. Beckett T.L. Studzinski C.M. Tomic J.L. Glabe C.G. Murphy M.P. Amyloid-β peptide and oligomers in the brain and cerebrospinal fluid of aged canines.J Alzheimers Dis. 2010; 20: 637-646Crossref PubMed Scopus (60) Google Scholar To determine the total amount of Aβ, capture was performed using monoclonal antibody Ab9 (against the amino-terminus of Aβ), and detection was performed using biotinylated 4G8 against Aβ17-24 (Covance), followed by horseradish peroxidase–conjugated NeutrAvidin (Pierce Biotechnology); Aβ40 was detected with Ab13.1.1, and Aβ42 was detected with 12F4 (Covance).38McGowan E. Pickford F. Kim J. Onstead L. Eriksen J. Yu C. Skipper L. Murphy M.P. Beard J. Das P. Jansen K. Delucia M. Lin W.L. Dolios G. Wang R. Eckman C.B. Dickson D.W. Hutton M. Hardy J. Golde T. Aβ42 is essential for parenchymal and vascular amyloid deposition in mice.Neuron. 2005; 47: 191-199Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar Alternatively, Aβ42 was captured using Ab2.1.3 and was detected with Ab9; alternative antibody combinations gave essentially identical results. For the measurement of APP or APP CTFs by ELISA, we used a similar procedure. Antibody 22C11 (Millipore) was used to capture full-length APP from the sample, which was then detected using biotinylated 6E10 against Aβ1–16 (Covance). The cleared sample was then transferred to a second plate, coated with affinity-purified antibody CT20, and raised against the last 20 amino acids of APP (see Supplemental Figure S2 at http://ajp.amjpathol.org). Captured CTFs were detected using either antibody 6E10 (for CTFβ) or 4G8 (for total CTFs). Oligomeric Aβ was measured using the single-site 4G8/4G8 sandwich ELISA.34Beckett T.L. Niedowicz D.M. Studzinski C.M. Weidner A.M. Webb R.L. Holler C.J. Ahmed R.R. LeVine 3rd, H. Murphy M.P. Effects of nonsteroidal anti-inflammatory drugs on amyloid-β pathology in mouse skeletal muscle.Neurobiol Dis. 2010; 39: 449-456Crossref PubMed Scopus (20) Google Scholar For RNA isolation, 100 mg of frozen tissue was homogenized using TRIzol reagent (Invitrogen) followed by phenol/chloroform extraction and ethanol precipitation, as per the manufacturer's instructions. RNeasy cleanup columns (Qiagen, Valencia, CA) were run for each sample. The reverse transcriptase reaction (iScript select cDNA synthesis kit; Bio-Rad Laboratories, Hercules, CA) used 1 μg of RNA, purified RNase H, MMLV reverse transcriptase, and a mixture of random hexamers and oligo dT primer, according to the manufacturer's instructions. Primers were designed with the following forward and reverse sequences, respectively: APP:
Alzheimer's disease (AD) involves multiple pathological processes in the brain, including increased inflammation and oxidative damage, as well as the accumulation of amyloid-β (Aβ) plaques. We hypothesized that a combinatorial therapeutic approach to target these multiple pathways may provide cognitive and neuropathological benefits for AD patients. To test this hypothesis, we used a canine model of human aging and AD. Aged dogs naturally develop learning and memory impairments, human-type Aβ deposits, and oxidative damage in the brain. Thus, 9 aged beagles (98-115 months) were treated with a medical food cocktail containing (1) an extract of turmeric containing 95% curcuminoids; (2) an extract of green tea containing 50% epigallocatechingallate; (3) N-acetyl cysteine; (4) R-alpha lipoic acid; and (5) an extract of black pepper containing 95% piperine. Nine similarly aged dogs served as placebo-treated controls. After 3 months of treatment, 13 dogs completed a variable distance landmark task used as a measure of spatial attention. As compared to placebo-treated animals, dogs receiving the medical food cocktail had significantly lower error scores (t11 = 4.3, p = 0.001) and were more accurate across all distances (F(1,9) = 20.7, p = 0.001), suggesting an overall improvement in spatial attention. Measures of visual discrimination learning, executive function and spatial memory, and levels of brain and cerebrospinal fluid Aβ were unaffected by the cocktail. Our results indicate that this medical food cocktail may be beneficial for improving spatial attention and motivation deficits associated with impaired cognition in aging and AD.
Many aging changes seem similar to those elicited by sleep-deprivation and psychosocial stress. Further, sleep architecture changes with age suggest an age-related loss of sleep. Here, we hypothesized that sleep deprivation in young subjects would elicit both stress and aging-like transcriptional responses.F344 rats were divided into control and sleep deprivation groups. Body weight, adrenal weight, corticosterone level and hippocampal CA1 transcriptional profiles were measured. A second group of animals was exposed to novel environment stress (NES), and their hippocampal transcriptional profiles measured. A third cohort exposed to control or SD was used to validate transcriptional results with Western blots. Microarray results were statistically contrasted with prior transcriptional studies. Microarray results pointed to sleep pressure signaling and macromolecular synthesis disruptions in the hippocampal CA1 region. Animals exposed to NES recapitulated nearly one third of the SD transcriptional profile. However, the SD-aging relationship was more complex. Compared to aging, SD profiles influenced a significant subset of genes. mRNA associated with neurogenesis and energy pathways showed agreement between aging and SD, while immune, glial, and macromolecular synthesis pathways showed SD profiles that opposed those seen in aging.We conclude that although NES and SD exert similar transcriptional changes, selective presynaptic release machinery and Homer1 expression changes are seen in SD. Among other changes, the marked decrease in Homer1 expression with age may represent an important divergence between young and aged brain response to SD. Based on this, it seems reasonable to conclude that therapeutic strategies designed to promote sleep in young subjects may have off-target effects in the aged. Finally, this work identifies presynaptic vesicular release and intercellular adhesion molecular signatures as novel therapeutic targets to counter effects of SD in young subjects.
Despite clinical evidence that thyroid hormone is essential for brain development before birth, effects of thyroid hormone on the fetal brain have been largely unexplored. One mechanism of thyroid hormone action is regulation of gene expression, because thyroid hormone receptors (TRs) are ligand-activated transcription factors. We used differential display to identify genes affected by acute T 4 administration to the dam before the onset of fetal thyroid function. Eight of the 11 genes that we identified were selectively expressed in brain areas known to contain TRs, indicating that these genes were directly regulated by thyroid hormone. Using in situ hybridization, we confirmed that the cortical expression of both neuroendocrine-specific protein (NSP) and Oct-1 was affected by changes in maternal thyroid status. Additionally, we demonstrated that both NSP and Oct-1 were expressed in the adult brain and that their responsiveness to thyroid hormone was retained. These data are the first to identify thyroid hormone-responsive genes in the fetal brain.
Polychlorinated biphenyls (PCBs) are a class of industrial compounds consisting of paired phenyl rings with various degrees of chlorination. They are now ubiquitous, persistent environmental contaminants that are routinely found in samples of human and animal tissues and are known to affect brain development. The effects of PCBs on brain development may be attributable, at least in part, to their ability to reduce circulating levels of thyroid hormone. However, the developmental effects of PCB exposure are not fully consistent with hypothyroidism. Because some individual PCB congeners interact strongly with various thyroid hormone binding proteins, several investigators have speculated that these congeners may be producing thyroid hormone-like effects on brain development. Therefore, we tested whether a mixture of PCBs, Aroclor 1254 (A1254), would produce an antithyroid or thyromimetic effect on the expression of known thyroid hormone-responsive genes in the developing brain. Pregnant female rats were fed various doses of A1254 (0, 1, 4, and 8 mg/kg) from gestational day 6 to weaning on postnatal day (P) 21. Pups derived from these dams were sampled on P5, P15, and P30. Total T4 was reduced by A1254 in a dose-dependent manner, but body weight of the pups or dams was not affected. The expression of RC3/Neurogranin and myelin basic protein was not affected by A1254 on P5 or P30. However, on P15, RC3/Neurogranin was elevated by A1254 in a dose-dependent manner, and myelin basic protein expression followed this general pattern. These data clearly demonstrate that the developmental effects of PCB exposure are not simply a function of PCB-induced hypothyroidism.
