Effects of Alzheimer's Disease on Different Cortical Layers

8496 • The Journal of Neuroscience, August 8, 2007 • 27(32):8496–8504 Neurobiology of Disease Effects of Alzheimer’s Disease on Different Cortical Layers: The Role of Intrinsic Differences in A␤ Susceptibility Rita R. Romito-DiGiacomo,1 Harry Menegay,3 Samantha A. Cicero,2 and Karl Herrup1 1Department of Neuroscience, Alzheimer Research Laboratory, and 2Department of Pharmacology, Case School of Medicine, Cleveland, Ohio 44106, and 3University Memory and Aging Center, Fairhill Center, Cleveland, Ohio 44120 Alzheimer’s disease is late life dementia associated with significant neurodegeneration in both cortical and subcortical regions. During the ϳ10 year course of the disease, neurons are lost in a progressive pattern that is relatively consistent among individuals. One example of this is the progression of disease pathology found in both the neocortex and archicortex. In these structures, the earliest problems can be found in superficial cortical layers (II–IV), whereas later the disease advances to involve the deeper cortical layers (V–VI). It is unclear whether these apparent differences in sensitivity are intrinsic to the neurons or imposed by external factors such as the pattern of connections. We used ␤-amyloid (A␤) peptide treatment of cultured mouse neurons as our model system. We show first that, as in hippocampus,dissociatedculturesofembryoniccorticalneuronsarebiasedtowardthesurvivalofcellsthatwerefinishingdivisioninthe ventricular zone at the time of harvest. Thus, embryonic day 13.5 (E13.5) cultures contain primarily deep-layer neurons whereas E16.5 cultures contain cells destined for upper layers. We use this cell-type specific segregation to our advantage and show, using both differences in gene expression profiles and A␤ survival curves, that deeper layer neurons are significantly more resistant to the toxic effects of A␤ than are cells from the more superficial strata. This suggests that an intrinsic underlying biology drives at least part of the AD progression pattern and that the time of harvest is a crucial variable in the interpretation of any cortical culture experiment. Key words: expression array; cell culture; rodent; neocortex; A␤ peptide; neuronal death Introduction erty of the neurons themselves or one imposed on the affected Alzheimer’s disease (AD) is the most common dementia of late neurons by virtue of their afferent or efferent connections. We life. Accompanying the neurological signs is a multidimensional sought to address this question in cell culture. pathology involving abnormal deposits of ␤-amyloid (A␤) pep- The neurons of the mammalian cerebral cortex are generated tides, hyperphosphorylated tau, atrophy of neurites and a pro- in a complex pattern of migration and settling with layers V and gressive loss of synapses, and, ultimately, loss of neuronal cell VI generated early [embryonic day 13 (E13) and E14 in mouse] bodies. Limbic structures are heavily affected (amygdala, ento- and layers II and III are generated later (ϳE16) (Takahashi et al., rhinal cortex, and hippocampus), but all regions of the brain 1993, 1995, 1999). Tangential migrants from the ganglionic em- seem to register some level of degenerative change. inence that give rise to the GABAergic neurons are mostly, but From the standpoint of understanding disease mechanism, it not completely present by E16 as well (Anderson et al., 1999; is intriguing that the progression of the disease is not uniform. Lavdas et al., 1999). Taking advantage of this invariant sequence, Limbic regions, in particular the perihippocampal formation, are we began a series of experiments aimed at determining the differ- affected first. Even within a single region, there appears to be a ential responsiveness of neocortical cells to the degenerative pro- regional progression. Detailed quantitative studies are not nu- cesses induced by the toxic effects of the A␤ peptide, a major merous, but the disease appears to involve the more superficial pathogenic agent in AD. We reasoned that, if we compared cul- layers of the cortex (layer II) and, to a lesser extent, layer IV, tures harvested on E13 with those harvested on E16, we could before progressing to involve all layers (Hyman et al., 1984; compare the responses of deep-layer cells (E13 harvest) with the Gomez-Isla et al., 1996). Human pathological studies are by their total population of the cortex (E16 harvest), allowing us to deter- nature descriptive, but this pattern of progression raises the mine whether the differential sensitivity of the different cortical mechanistic question of whether the pattern is an intrinsic prop- layers (Gomez-Isla et al., 1996) was apparent in our tissue-culture model. Received Oct. 4, 2006; revised June 20, 2007; accepted June 20, 2007. We report here that there is a clear intrinsic difference in A␤ This work was supported by the National Institutes of Health Grant NS20591 and a major gift from Coins for Alzheimer’s Research Trust. Funds were also obtained from the Blanchette Hooker Rockefeller Fund. We thank Drs. susceptibility between the deeper, more resistant layer V/VI neu- Randall York and Jianmin Chen for helpful discussions during the preparation of this manuscript. rons and superficial, more sensitive layer II/III neurons. In pur- Correspondence should be addressed to Karl Herrup at the above address. E-mail: [email protected]. suit of these studies, we have relearned the 30-year-old lessons of S.A.Cicero’spresentaddress:St.JudeChildren’sResearchHospital,DepartmentofDevelopmentalNeurobiology, Banker and Cowan (1977). Under standard conditions of disso- 332 North Lauderdale, Danny Thomas Research Center D2031, Memphis, TN 38105. DOI:10.1523/JNEUROSCI.1008-07.2007 ciation and plating, the harvest of rodent cerebral cortical neu- Copyright © 2007 Society for Neuroscience 0270-6474/07/278496-09$15.00/0 rons is heavily biased toward recovery of cells that are just com- Romito-DiGiacomo et al. • Layer-Specific Amyloid Vulnerability J. Neurosci., August 8, 2007 • 27(32):8496–8504 • 8497 University Institutional Animal Care and Use Committee considered and approved all procedures. Cortical cultures. Pregnant dams were killed by cervical dislocation and embryos were dissected from the uterine horns on ice-cold PBS/ glucose (1 mg/ml) as described previously (Ci- cero and Herrup, 2005). Briefly, brains were dissected and cortices removed and collected in ice-cold PBS/glucose. The cortices were then stripped of the meninges, minced, and trypsinized at 37°C for 10–15 min. The tissue was then transferred to a conical tube containing DMEM plus 10% FCS to inactivate the tryp- sin. The tissue was then transferred to a conical tube containing Neurobasal media supple- mented with B-27, penicillin/streptomycin, and2mML-glutamine (Invitrogen, Carlsbad, CA). Tissue was then triturated and live cell number was determined by staining with Trypan blue. Cells were then plated at a density of 50,000 cells/well in 24-well plates containing poly-L-lysine (0.05 mg/ml; P-4707; Sigma, St. Louis, MO)-coated glass coverslips, or at a density of 2.6 million cells for 100 mm poly-L- lysine-coated plates. Cells were then incubated for a period of 4–7 d in vitro (DIV) in Neuro- basal medium. Cultures to be treated with amyloid-␤ peptide were first grown undis- turbed for 5 d and then treated. The A␤ peptide was purchased from American Peptide (Sunny- vale, CA) as a dry powder and resuspended to a concentration of 2 mM using sterile water. The A␤ solution was allowed to sit at room temper- ature for ϳ1 h before vortexing aliquoting and freezing at Ϫ20°C. Individual aliquots were fibrillized by incubating them at 37°C 5 d before use; any remaining fibrillized A␤ was stored at 4°C. Figure1. BrdUincorporationandquantificationinE15.5embryos.A,E15.5corticalcultureslabeledwithBrdUfor2h(a)or48h Cell counts. Cell death in response to A␤ (b) before being killed. Cultures were stained with TuJ1 (red) and BrdU (green). Corresponding sections were also stained with 1–42 treatment was determined in E13.5–E17.5 cul- BrdU (green) and Tbr-1 (red) and show BrdU incorporation restricted to the ventricular zone in the 2 h injected cortex (c) whereas tures grown for 5 DIV. Briefly, cultures were the 48 h injection shows labeled cells throughout the cortex (d). B, Summary of the quantification of BrdU incorporation in E15.5 treated with A␤ at concentrations of 0, 0.3, cultures killed either 2 or 48 h after injection. Cultures were stained with BrdU and TuJ1 and counts were done on double-positive 1–42 1, 3, 10, and 30 ␮M for 72 h. After treatment, cells. The graph shows more intensely stained cells in the 2 h injected cultures and more diffusely stained cells in the 48 h injected cells were stained using the Live/Dead Viability/ cultures.Despitetheexpecteddifferenceinintensity,therelativenumberoftotalBrdUpositivecellsisverysimilar,consistentwith Cytotoxicity kit from Invitrogen and cell loss the belief that only cells in the ventricular zone survive culturing. C, D, The in vivo staining pattern further confirms this belief was determined by counts. Each age of culture showing a large number of BrdU-positive cells throughout the cortex of a E13/15 (C) embryo when compared with an E15.5 was repeated on three separate experiments; in embryo injected with BrdU 2 h earlier (E15 ϩ2; D). We would expect the counts in B to reflect this difference if all cortical layers each experiment, nine fields were counted in survived culturing. Scale bars: (in Aa) Aa, Ab,50␮m; (in Ac) Ac, Ad, 100 ␮m. VZ, Ventricular zone; IZ, intermediate zone; SP, each of three wells for each treatment concen- subplate; CP, cortical plate; MZ, marginal zone; P, pia. tration (27 total fields/treatment/experiment) at 40ϫ magnification. Total cell numbers were pleting neurogenesis. The findings offer new strategies for combined and averaged to generate a response curve for all ages tested. approaching the analysis of cortical neuron behavior and also Percent loss was calculated from untreated controls. For T-box protein ␤ suggest caution in the interpretation of the existing literature on (Tbr-1) survival, E13.5 and E16.5 cultures were treated with A 1–42 for A␤ responsiveness of cortical cultures.

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