Stephen D Ginsberg, Ph.D.

Microarray analysis of CA1 pyramidal neurons in a mouse model of tauopathy reveals progressive synaptic dysfunction
Alldred, Melissa J; Duff, Karen E; Ginsberg, Stephen D
2012 Feb;45(2):751-762, Neurobiology of disease
The hTau mouse model of tauopathy was utilized to assess gene expression changes in vulnerable hippocampal CA1 neurons. CA1 pyramidal neurons were microaspirated via laser capture microdissection followed by RNA amplification in combination with custom-designed microarray analysis and qPCR validation in hTau mice and nontransgenic (ntg) littermates aged 11-14months. Statistical analysis revealed ~8% of all the genes on the array platform were dysregulated, with notable downregulation of several synaptic-related markers including synaptophysin (Syp), synaptojanin, and synaptobrevin, among others. Downregulation was also observed for select glutamate receptors (GluRs), Psd-95, TrkB, and several protein phosphatase subunits. In contrast, upregulation of tau isoforms and a calpain subunit were found. Microarray assessment of synaptic-related markers in a separate cohort of hTau mice at 7-8months of age indicated only a few alterations compared to the 11-14month cohort, suggesting progressive synaptic dysfunction occurs as tau accumulates in CA1 pyramidal neurons. An assessment of SYP and PSD-95 expression was performed in the hippocampal CA1 sector of hTau and ntg mice via confocal laser scanning microscopy along with hippocampal immunoblot analysis for protein-based validation of selected microarray observations. Results indicate significant decreases in SYP-immunoreactive and PSD-95-immunoreactive puncta as well as downregulation of SYP-immunoreactive and PSD-95-immunoreactive band intensity in hTau mice compared to age-matched ntg littermates. In summary, the high prevalence of downregulation of synaptic-related genes indicates that the moderately aged hTau mouse may be a model of tau-induced synaptodegeneration, and has profound effects on how we perceive progressive tau pathology affecting synaptic transmission in AD
— id: 149951, year: 2012, vol: 45, page: 751, stat: Journal Article,

Gene expression levels assessed by CA1 pyramidal neuron and regional hippocampal dissections in Alzheimer's disease
Ginsberg, Stephen D; Alldred, Melissa J; Che, Shaoli
2012 Jan;45(1):99-107, Neurobiology of disease
To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer's disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific neurons and intrinsic circuits will likely yield informative gene expression profile data that may be subthreshold and/or underrepresented in regional studies with an admixture of cell types
— id: 141967, year: 2012, vol: 45, page: 99, stat: Journal Article,

Expression profiling in neuropsychiatric disorders: Emphasis on glutamate receptors in bipolar disorder
Ginsberg, Stephen D; Hemby, Scott E; Smiley, John F
2012 Feb;100(4):705-711, Pharmacology biochemistry & behavior
Functional genomics and proteomics approaches are being employed to evaluate gene and encoded protein expression changes with the tacit goal to find novel targets for drug discovery. Genome-wide association studies (GWAS) have attempted to identify valid candidate genes through single nucleotide polymorphism (SNP) analysis. Furthermore, microarray analysis of gene expression in brain regions and discrete cell populations has enabled the simultaneous quantitative assessment of relevant genes. The ability to associate gene expression changes with neuropsychiatric disorders, including bipolar disorder (BP), and their response to therapeutic drugs provides a novel means for pharmacotherapeutic interventions. This review summarizes gene and pathway targets that have been identified in GWAS studies and expression profiling of human postmortem brain in BP, with an emphasis on glutamate receptors (GluRs). Although functional genomic assessment of BP is in its infancy, results to date point towards a dysregulation of GluRs that bear some similarity to schizophrenia (SZ), although the pattern is complex, and likely to be more complementary than overlapping. The importance of single population expression profiling of specific neurons and intrinsic circuits is emphasized, as this approach provides informative gene expression profile data that may be underappreciated in regional studies with admixed neuronal and non-neuronal cell types
— id: 149796, year: 2012, vol: 100, page: 705, stat: Journal Article,

Mild cognitive impairment: pathology and mechanisms
Mufson, Elliott J.; Binder, Lester; Counts, Scott E.; DeKosky, Steven T.; deToledo-Morrell, Leyla; Ginsberg, Stephen D.; Ikonomovic, Milos D.; Perez, Sylvia E.; Scheff, Stephen W.
2012 JAN ;123(1):13-30, Acta neuropathologica
Mild cognitive impairment (MCI) is rapidly becoming one of the most common clinical manifestations affecting the elderly. The pathologic and molecular substrate of people diagnosed with MCI is not well established. Since MCI is a human specific disorder and neither the clinical nor the neuropathological course appears to follow a direct linear path, it is imperative to characterize neuropathology changes in the brains of people who came to autopsy with a well-characterized clinical diagnosis of MCI. Herein, we discuss findings derived from clinical pathologic studies of autopsy cases who died with a clinical diagnosis of MCI. The heterogeneity of clinical MCI imparts significant challenges to any review of this subject. The pathologic substrate of MCI is equally complex and must take into account not only conventional plaque and tangle pathology but also a wide range of cellular, biochemical and molecular deficits, many of which relate to cognitive decline as well as compensatory responses to the progressive disease process. The multifaceted nature of the neuronal disconnection syndrome associated with MCI suggests that there is no single event which precipitates this prodromal stage of AD. In fact, it can be argued that neuronal degeneration initiated at different levels of the central nervous system drives cognitive decline as a final common pathway at this stage of the dementing disease process
— id: 150798, year: 2012, vol: 123, page: 13, stat: Journal Article,

Gender differences in neurotrophin and glutamate receptor expression in cholinergic nucleus basalis neurons during the progression of Alzheimer's disease
Counts, Scott E.; Che, Shaoli; Ginsberg, Stephen D.; Mufson, Elliott J.
2011 OCT ;42(2):111-117, Journal of chemical neuroanatomy
The higher incidence rate of Alzheimer's disease (AD) in elderly women indicates that gender plays a role in AD pathogenesis. Evidence from clinical and pharmacologic studies, neuropathological examinations, and models of hormone replacement therapy suggest that cholinergic basal forebrain (CBF) cortical projection neurons within the nucleus basalis (NB), which mediate memory and attention and degenerate in AD, may be preferentially vulnerable in elderly women compared to men. CBF neurons depend on nerve growth factor (NGF) and their cognate receptors (trkA and p75(NTR)) for their survival and maintenance. We recently demonstrated a shift in the balance of NGF and its receptors toward cell death mechanisms during the progression of AD. To address whether gender affects NGF signaling system expression within the CBF, we used single cell RNA amplification and custom microarray technologies to compare gene expression profiles of single cholinergic NB neurons in tissue specimens from male and female members of the Religious Orders Study who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. p75(NTR) expression within male cholinergic NB neurons was unchanged across clinical diagnosis, whereas p75(NTR) mRNA levels in female NB neurons exhibited a similar to 40% reduction in AD compared to NCI. Male AD subjects displayed a similar to 45% reduction in trkA mRNA levels within NB neurons compared to NCI and MCI. In contrast, NB neuronal trkA expression in females was reduced similar to 50% in both MCI and AD compared to NCI. Reduced trkA mRNA levels were associated with poorer global cognitive performance and higher Braak scores in the female subjects. In addition, we found a female-selective reduction in GluR2 AMPA glutamate receptor subunit expression in NB neurons in AD. These data suggest that cholinergic NB neurons in females may be at greater risk for degeneration during the progression of AD and support the concept of gender-specific therapeutic interventions during the preclinical stages of the disease. Published by Elsevier B.V
— id: 138469, year: 2011, vol: 42, page: 111, stat: Journal Article,

Differential regulation of catechol-O-methyltransferase expression in a mouse model of aggression
Ginsberg SD; Che S; Hashim A; Zavadil J; Cancro R; Lee SH; Petkova E; Sershen HW; Volavka J
2011 Nov;216(4):347-356, Brain structure & function
This study was designed to understand molecular and cellular mechanisms underlying aggressive behaviors in mice exposed to repeated interactions in their homecage with conspecifics. A resident-intruder procedure was employed whereby two males were allowed to interact for 10 min trials, and aggressive and/or submissive behaviors (e.g., degree of attacking, biting, chasing, grooming, rearing, or upright posture) were assessed. Following 10 days of behavioral trials, brains were removed and dissected into specific regions including the cerebellum, frontal cortex, hippocampus, midbrain, pons, and striatum. Gene expression analysis was performed using real-time quantitative polymerase-chain reaction (qPCR) for catechol-O-methyltransferase (COMT) and tyrosine hydroxylase (TH). Compared to naive control mice, significant up regulation of COMT expression of residents was observed in the cerebellum, frontal cortex, hippocampus, midbrain, and striatum; in all of these brain regions the COMT expression of residents was also significantly higher than that of intruders. The intruders also had a significant down regulation (compared to naive control mice) within the hippocampus, indicating a selective decrease in COMT expression in the hippocampus of submissive subjects. Immunoblot analysis confirmed COMT up regulation in the midbrain and hippocampus of residents and down regulation in intruders. qPCR analysis of TH expression indicated significant up regulation in the midbrain of residents and concomitant down regulation in intruders. These findings implicate regionally- and behaviorally-specific regulation of COMT and TH expression in aggressive and submissive behaviors. Additional molecular and cellular characterization of COMT, TH, and other potential targets is warranted within this animal model of aggression
— id: 137055, year: 2011, vol: 216, page: 347, stat: Journal Article,

Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease
Ginsberg, Stephen D; Mufson, Elliott J; Alldred, Melissa J; Counts, Scott E; Wuu, Joanne; Nixon, Ralph A; Che, Shaoli
2011 Oct;42(2):102-110, Journal of chemical neuroanatomy
Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer's disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD
— id: 136996, year: 2011, vol: 42, page: 102, stat: Journal Article,

Vacuolar pathology in the median eminence of the hypothalamus after hyponatremia
Levine, Seymour; Saltzman, Arthur; Ginsberg, Stephen D
2011 Feb;70(2):151-156, Journal of neuropathology & experimental neurology
The median eminence of the hypothalamus is an important conduit by which neurosecretory hormones from hypothalamic nuclei are delivered to the pars nervosa (neural lobe) of the pituitary en route tothe bloodstream. Dilutional hyponatremia was produced in adult ratsto determine the effect on the morphology of the median eminence of the hypothalamus. Hyponatremia was caused by reducing electrolyte and organic osmolyte reserves to block the excretion of water through delivery of the nephrotoxin mercuric chloride (HgCl2). Histological examination of the brain 1 day after a hyponatremic insult revealed vacuolation within the median eminence of the hypothalamus. No other lesions were found in other parts of the brain after hyponatremia. The hyponatremic lesion consisted of a band of closely packed vacuoles that crossed the floor of the third ventricle. Vacuoles associated with hyponatremia were predominantly in the subependymal, fiber, reticular, and palisade layers of the median eminence. Vacuolation was not observed in the tanycyte layer of the median eminence. This study indicates that the median eminence is a potentially vulnerable site in human hyponatremic conditions that should be evaluated further in relevant animal models
— id: 124107, year: 2011, vol: 70, page: 151, stat: Journal Article,

A genotype resource for postmortem brain samples from the Autism Tissue Program
Wintle, Richard F; Lionel, Anath C; Hu, Pingzhao; Ginsberg, Stephen D; Pinto, Dalila; Thiruvahindrapduram, Bhooma; Wei, John; Marshall, Christian R; Pickett, Jane; Cook, Edwin H; Scherer, Stephen W
2011 Apr;4(2):89-97, Autism research
The Autism Tissue Program (ATP), a science program of Autism Speaks, provides researchers with access to well-characterized postmortem brain tissues. Researchers access these tissues through a peer-reviewed, project-based approval process, and obtain related clinical information from a secure, online informatics portal. However, few of these samples have DNA banked from other sources (such as a blood sample from the same individual), hindering genotype-phenotype correlation and interpretation of gene expression data derived from the banked brain tissue. Here, we describe an initiative to extract DNA from Brodmann Area 19, and genotype these samples using both the Affymetrix Genome-Wide Human SNP Array 6.0 and the Illumina Human1M-Duo DNA Analysis BeadChip genome-wide microarray technologies. We additionally verify reported gender, and infer ethnic background from the single nucleotide polymorphism data. We have also used a rigorous, multiple algorithm approach to identify genomic copy number variation (CNV) from these array data. Following an initial proof of principle study using two samples, 52 experimental samples, consisting of 27 subjects with confirmed or suspected autism and related disorders, 5 subjects with cytogenetically visible duplications of 15q, 2 with epilepsy and 18 age-matched normal controls were processed, yielding high-quality genotype data in all cases. The genotype and CNV data are provided via the ATP informatics portal as a resource for the autism research community
— id: 134278, year: 2011, vol: 4, page: 89, stat: Journal Article,

Sex- and brain region-specific acceleration of beta-amyloidogenesis following behavioral stress in a mouse model of Alzheimer's disease
Devi, Latha; Alldred, Melissa J; Ginsberg, Stephen D; Ohno, Masuo
2010 ;3:34-34, Mol Brain
BACKGROUND: It is hypothesized that complex interactions between multiple environmental factors and genetic factors are implicated in sporadic Alzheimer's disease (AD); however, the underlying mechanisms are poorly understood. Importantly, recent evidence reveals that expression and activity levels of the beta-site APP cleaving enzyme 1 (BACE1), which initiates amyloid-beta (Abeta) production, are elevated in AD brains. In this study, we investigated a molecular mechanism by which sex and stress interactions may accelerate beta-amyloidogenesis and contribute to sporadic AD. RESULTS: We applied 5-day restraint stress (6 h/day) to the male and female 5XFAD transgenic mouse model of AD at the pre-pathological stage of disease, which showed little amyloid deposition under non-stressed control conditions. Exposure to the relatively brief behavioral stress increased levels of neurotoxic Abeta42 peptides, the beta-secretase-cleaved C-terminal fragment (C99) and plaque burden in the hippocampus of female 5XFAD mice but not in that of male 5XFAD mice. In contrast, significant changes in the parameters of beta-amyloidosis were not observed in the cerebral cortex of stressed male or female 5XFAD mice. We found that this sex- and brain region-specific acceleration of beta-amyloidosis was accounted for by elevations in BACE1 and APP levels in response to adverse stress. Furthermore, not only BACE1 mRNA but also phosphorylation of the translation initiation factor eIF2alpha (a proposed mediator of the post-transcriptional upregulation of BACE1) was elevated in the hippocampus of stressed female 5XFAD mice. CONCLUSIONS: Our results suggest that the higher prevalence of sporadic AD in women may be attributable to the vulnerability of female brains (especially, the hippocampus) to stressful events, which alter APP processing to favor the beta-amyloidogenesis through the transcriptional and translational upregulation of BACE1 combined with elevations in its substrate APP.
— id: 155560, year: 2010, vol: 3, page: 34, stat: Journal Article,

Alterations in discrete glutamate receptor subunits in adult mouse dentate gyrus granule cells following perforant path transection
Ginsberg, Stephen D
2010 Aug;397(8):3349-3358, Analytical & bioanalytical chemistry
Custom-designed microarray analysis was utilized to evaluate expression levels of glutamate receptors (GluRs) and GluR-interacting protein genes within isolated dentate gyrus granule cells following axotomy of the principal input, the perforant path (PP). Dentate gyrus granule cells were evaluated by microdissection via laser capture microdissection, terminal continuation RNA amplification, and microarray analysis following unilateral PP transections at seven time points. Expression profiles garnered from granule cells on the side ipsilateral to PP transections were compared and contrasted with naive subjects and mice subjected to unilateral occipital cortex lesions. Selected microarray observations were validated by real-time quantitative PCR analysis. Postlesion time-dependent alterations in specific alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, kainate receptors, N-methyl-D: -aspartate (NMDA) receptors, and GluR-interacting protein genes were found across the time course of the study, suggesting a neuroplasticity response associated with the transsynaptic granule cell alterations following axotomy of incoming PP terminals
— id: 111355, year: 2010, vol: 397, page: 3349, stat: Journal Article,

Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression
Ginsberg, Stephen D; Alldred, Melissa J; Counts, Scott E; Cataldo, Anne M; Neve, Rachael L; Jiang, Ying; Wuu, Joanne; Chao, Moses V; Mufson, Elliott J; Nixon, Ralph A; Che, Shaoli
2010 Nov 15;68(10):885-893, Biological psychiatry
BACKGROUND: Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimer's disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis. METHODS: Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection was performed using postmortem brain tissue. Validation was achieved using real-time quantitative polymerase chain reaction and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via small interference RNA. RESULTS: Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively upregulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal quantitative polymerase chain reaction and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the downregulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB downregulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD. CONCLUSIONS: These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long-term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression
— id: 114169, year: 2010, vol: 68, page: 885, stat: Journal Article,

Regional Selectivity of rab5 and rab7 Protein Upregulation in Mild Cognitive Impairment and Alzheimer's Disease
Ginsberg, Stephen D; Mufson, Elliott J; Counts, Scott E; Wuu, Joanne; Alldred, Melissa J; Nixon, Ralph A; Che, Shaoli
2010 ;22(2):631-639, Journal of Alzheimer's Disease
Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR experiments revealed an upregulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of people with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Results indicate selective upregulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD
— id: 114587, year: 2010, vol: 22, page: 631, stat: Journal Article,

Controlled enzymatic production of astrocytic hydrogen peroxide protects neurons from oxidative stress via an Nrf2-independent pathway
Haskew-Layton, Renee E; Payappilly, Jimmy B; Smirnova, Natalya A; Ma, Thong C; Chan, Kelvin K; Murphy, Timothy H; Guo, Hengchang; Langley, Brett; Sultana, Rukhsana; Butterfield, D Allan; Santagata, Sandro; Alldred, Melissa J; Gazaryan, Irina G; Bell, George W; Ginsberg, Stephen D; Ratan, Rajiv R
2010 Oct 5;107(40):17385-17390, Proceedings of the National Academy of Sciences of the United States of America
Neurons rely on their metabolic coupling with astrocytes to combat oxidative stress. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) appears important for astrocyte-dependent neuroprotection from oxidative insults. Indeed, Nrf2 activators are effective in stroke, Parkinson disease, and Huntington disease models. However, key endogenous signals that initiate adaptive neuroprotective cascades in astrocytes, including activation of Nrf2-mediated gene expression, remain unclear. Hydrogen peroxide (H(2)O(2)) plays an important role in cell signaling and is an attractive candidate mediator of adaptive responses in astrocytes. Here we determine (i) the significance of H(2)O(2) in promoting astrocyte-dependent neuroprotection from oxidative stress, and (ii) the relevance of H(2)O(2) in inducing astrocytic Nrf2 activation. To control the duration and level of cytoplasmic H(2)O(2) production in astrocytes cocultured with neurons, we heterologously expressed the H(2)O(2)-producing enzyme Rhodotorula gracilis D-amino acid oxidase (rgDAAO) selectively in astrocytes. Exposure of rgDAAO-astrocytes to D-alanine lead to the concentration-dependent generation of H(2)O(2). Seven hours of low-level H(2)O(2) production ( approximately 3.7 nmol.min.mg protein) in astrocytes protected neurons from oxidative stress, but higher levels ( approximately 130 nmol.min.mg protein) were neurotoxic. Neuroprotection occurred without direct neuronal exposure to astrocyte-derived H(2)O(2), suggesting a mechanism specific to astrocytic intracellular signaling. Nrf2 activation mimicked the effect of astrocytic H(2)O(2) yet H(2)O(2)-induced protection was independent of Nrf2. Astrocytic protein tyrosine phosphatase inhibition also protected neurons from oxidative death, representing a plausible mechanism for H(2)O(2)-induced neuroprotection. These findings demonstrate the utility of rgDAAO for spatially and temporally controlling intracellular H(2)O(2) concentrations to uncover unique astrocyte-dependent neuroprotective mechanisms
— id: 135352, year: 2010, vol: 107, page: 17385, stat: Journal Article,

Alzheimer's-related endosome dysfunction in Down syndrome is Abeta-independent but requires APP and is reversed by BACE-1 inhibition
Jiang, Ying; Mullaney, Kerry A; Peterhoff, Corrinne M; Che, Shaoli; Schmidt, Stephen D; Boyer-Boiteau, Anne; Ginsberg, Stephen D; Cataldo, Anne M; Mathews, Paul M; Nixon, Ralph A
2010 Jan 26;107(4):1630-1635, Proceedings of the National Academy of Sciences of the United States of America
An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics
— id: 126490, year: 2010, vol: 107, page: 1630, stat: Journal Article,

Cystatin C rescues degenerating neurons in a cystatin B-knockout mouse model of progressive myoclonus epilepsy
Kaur, Gurjinder; Mohan, Panaiyur; Pawlik, Monika; DeRosa, Steven; Fajiculay, Jay; Che, Shaoli; Grubb, Anders; Ginsberg, Stephen D; Nixon, Ralph A; Levy, Efrat
2010 Nov;177(5):2256-2267, American journal of pathology
In vitro studies have shown that cystatin C (CysC) is neuroprotective. Here we demonstrate that CysC is neuroprotective in vivo, in a mouse model of the inherited neurodegenerative disorder, progressive myoclonic epilepsy type 1 (EPM1). Loss-of-function mutations in the cystatin B (CysB) gene, an intracellular cysteine protease inhibitor, lead to this human disease. A CysB-knockout (CysBKO) mouse model develops symptoms that mimic EPM1. CysB deficiency in these mice results in enhanced cathepsin B and D activities, indicating lysosomal dysfunction. We show that expression of CysC is enhanced in the brains of CysBKO mice. Crossbreeding of CysBKO mice with either CysC-overexpressing transgenic mice or CysC-knockout mice demonstrates that clinical symptoms and neuropathologies, including motor coordination disorder, cerebellar atrophy, neuronal loss in the cerebellum and cerebral cortex, and gliosis caused by CysB deficiency, are rescued by CysC overexpression and exacerbated by CysC deficiency. Thus, CysC effectively rescues the CysB loss-of-function mutations, facilitating the reversal of pathophysiological changes and suggesting a novel therapeutic intervention for patients with EPM1 and other neurodegenerative disorders
— id: 126484, year: 2010, vol: 177, page: 2256, stat: Journal Article,

Mitotic figures in the median eminence of the hypothalamus
Levine, Seymour; Saltzman, Arthur; Ginsberg, Stephen D
2010 Nov;35(11):1743-1746, Neurochemical research
The median eminence of the hypothalamus is part of the avenue by which neurosecreted hormones from the hypothalamic nuclei reach the pars nervosa (neural lobe) of the pituitary and eventually the bloodstream. Lithium treatment and osmotic stress increases the transport of neurosecretory hormones to the pituitary in the adult rat. Specialized astrocytes termed pituicytes in the pars nervosa of the pituitary participate in the secretory process and also develop considerable mitotic activity. The present work reveals similar mitotic figures in cells within the median eminence following 3 days of lithium treatment. The location and appearance of these mitoses add to the evidence that pituicytes are present in the median eminence. Moreover, mitoses occur within the ependymal (tanycyte) layer of the median eminence. Thus, the present results suggest that the tanycyte layer may contain pituicytes, indicating that the hypothalamus possesses specialized cells for modulating neurosecretion in response to osmotic challenges
— id: 113941, year: 2010, vol: 35, page: 1743, stat: Journal Article,

Terminal continuation (TC) RNA amplification without second strand synthesis
Alldred, Melissa J; Che, Shaoli; Ginsberg, Stephen D
2009 Mar 15;177(2):381-385, Journal of neuroscience methods
Terminal continuation (TC) RNA amplification was developed originally to reproducibly and inexpensively amplify RNA. The TC RNA amplification method has been improved further by obviating second strand DNA synthesis, a cost-effective protocol that takes less time to perform with fewer manipulations required for RNA amplification. Results demonstrate that TC RNA amplification without second strand synthesis does not differ from the original protocol using RNA harvested from mouse brain and from hippocampal neurons obtained via laser capture microdissection from postmortem human brains. The modified TC RNA amplification method can discriminate single cell gene expression profiles between normal control and Alzheimer's disease hippocampal neurons indistinguishable from the original protocol. Thus, TC RNA amplification without second strand synthesis is a reproducible, time- and cost-effective method for RNA amplification from minute amounts of input RNA, and is compatible with microaspiration strategies and subsequent microarray analysis as well as quantitative real-time PCR
— id: 105217, year: 2009, vol: 177, page: 381, stat: Journal Article,

Target identification for CNS diseases by transcriptional profiling
Altar, C Anthony; Vawter, Marquis P; Ginsberg, Stephen D
2009 Jan;34(1):18-54, Neuropsychopharmacology
Gene expression changes in neuropsychiatric and neurodegenerative disorders, and gene responses to therapeutic drugs, provide new ways to identify central nervous system (CNS) targets for drug discovery. This review summarizes gene and pathway targets replicated in expression profiling of human postmortem brain, animal models, and cell culture studies. Analysis of isolated human neurons implicates targets for Alzheimer's disease and the cognitive decline associated with normal aging and mild cognitive impairment. In addition to tau, amyloid-beta precursor protein, and amyloid-beta peptides (Abeta), these targets include all three high-affinity neurotrophin receptors and the fibroblast growth factor (FGF) system, synapse markers, glutamate receptors (GluRs) and transporters, and dopamine (DA) receptors, particularly the D2 subtype. Gene-based candidates for Parkinson's disease (PD) include the ubiquitin-proteosome system, scavengers of reactive oxygen species, brain-derived neurotrophic factor (BDNF), its receptor, TrkB, and downstream target early growth response 1, Nurr-1, and signaling through protein kinase C and RAS pathways. Increasing variability and decreases in brain mRNA production from middle age to old age suggest that cognitive impairments during normal aging may be addressed by drugs that restore antioxidant, DNA repair, and synaptic functions including those of DA to levels of younger adults. Studies in schizophrenia identify robust decreases in genes for GABA function, including glutamic acid decarboxylase, HINT1, glutamate transport and GluRs, BDNF and TrkB, numerous 14-3-3 protein family members, and decreases in genes for CNS synaptic and metabolic functions, particularly glycolysis and ATP generation. Many of these metabolic genes are increased by insulin and muscarinic agonism, both of which are therapeutic in psychosis. Differential genomic signals are relatively sparse in bipolar disorder, but include deficiencies in the expression of 14-3-3 protein members, implicating these chaperone proteins and the neurotransmitter pathways they support as possible drug targets. Brains from persons with major depressive disorder reveal decreased expression for genes in glutamate transport and metabolism, neurotrophic signaling (eg, FGF, BDNF and VGF), and MAP kinase pathways. Increases in these pathways in the brains of animals exposed to electroconvulsive shock and antidepressant treatments identify neurotrophic and angiogenic growth factors and second messenger stimulation as therapeutic approaches for the treatment of depression
— id: 133661, year: 2009, vol: 34, page: 18, stat: Journal Article,

In vivo MRI identifies cholinergic circuitry deficits in a Down syndrome model
Chen, Yuanxin; Dyakin, Victor V; Branch, Craig A; Ardekani, Babak; Yang, Dunsheng; Guilfoyle, David N; Peterson, Jesse; Peterhoff, Corrinne; Ginsberg, Stephen D; Cataldo, Anne M; Nixon, Ralph A
2009 Sep;30(9):1453-1465, Neurobiology of aging
In vivo quantitative magnetic resonance imaging (MRI) was employed to detect brain pathology and map its distribution within control, disomic mice (2N) and in Ts65Dn and Ts1Cje trisomy mice with features of human Down syndrome (DS). In Ts65Dn, but not Ts1Cje mice, transverse proton spin-spin (T(2)) relaxation time was selectively reduced in the medial septal nucleus (MSN) and in brain regions that receive cholinergic innervation from the MSN, including the hippocampus, cingulate cortex, and retrosplenial cortex. Basal forebrain cholinergic neurons (BFCNs) in the MSN, identified by choline acetyltransferase (ChAT) and nerve growth factor receptors p75(NTR) and TrkA immunolabeling were reduced in Ts65Dn brains and in situ acetylcholinesterase (AChE) activity was depleted distally along projecting cholinergic fibers, and selectively on pre- and postsynaptic profiles in these target areas. T(2) effects were negligible in Ts1Cje mice that are diploid for App and lack BFCN neuropathology, consistent with the suspected relationship of this pathology to increased App dosage. These results establish the utility of quantitative MRI in vivo for identifying Alzheimer's disease-relevant cholinergic changes in animal models of DS and characterizing the selective vulnerability of cholinergic neuron subpopulations
— id: 86660, year: 2009, vol: 30, page: 1453, stat: Journal Article,

Age-dependent dysregulation of brain amyloid precursor protein in the Ts65Dn Down syndrome mouse model
Choi, Jennifer H K; Berger, Jason D; Mazzella, Matthew J; Morales-Corraliza, Jose; Cataldo, Anne M; Nixon, Ralph A; Ginsberg, Stephen D; Levy, Efrat; Mathews, Paul M
2009 Sep;110(6):1818-1827, Journal of neurochemistry
Individuals with Down syndrome develop beta-amyloid deposition characteristic of early-onset Alzheimer's disease (AD) in mid-life, presumably because of an extra copy of the chromosome 21-located amyloid precursor protein (App) gene. App mRNA and APP metabolite levels were assessed in the brains of Ts65Dn mice, a mouse model of Down syndrome, using quantitative PCR, western blot analysis, immunoprecipitation, and ELISAs. In spite of the additional App gene copy, App mRNA, APP holoprotein, and all APP metabolite levels in the brains of 4-month-old trisomic mice were not increased compared with the levels seen in diploid littermate controls. However starting at 10 months of age, brain APP levels were increased proportional to the App gene dosage imbalance reflecting increased App message levels in Ts65Dn mice. Similar to APP levels, soluble amino-terminal fragments of APP (sAPPalpha and sAPPbeta) were increased in Ts65Dn mice compared with diploid mice at 12 months but not at 4 months of age. Brain levels of both Abeta40 and Abeta42 were not increased in Ts65Dn mice compared with diploid mice at all ages examined. Therefore, multiple mechanisms contribute to the regulation towards diploid levels of APP metabolites in the Ts65Dn mouse brain
— id: 126493, year: 2009, vol: 110, page: 1818, stat: Journal Article,

Galanin fiber hyperinnervation preserves neuroprotective gene expression in cholinergic basal forebrain neurons in Alzheimer's disease
Counts, Scott E; He, Bin; Che, Shaoli; Ginsberg, Stephen D; Mufson, Elliott J
2009 ;18(4):885-896, Journal of Alzheimer's Disease
Fibers containing galanin (GAL) hyperinnervate cholinergic basal forebrain (CBF) nucleus basalis neurons in late stage Alzheimer's disease (AD), yet the molecular consequences of this phenomenon are unknown. To determine whether GAL alters the expression of genes critical to CBF cell survival in AD, single cell microarray analysis was used to determine mRNA levels within nucleus basalis neurons lacking GAL innervation from subjects who died with a clinical diagnosis of no cognitive impairment (NCI) compared to nucleus basalis neurons from AD cases either lacking GAL hyperinnervation (AD/GAL-) or those displaying prominent GAL hyperinnervation (AD/GAL+). Levels of mRNAs encoding putatively neuroprotective proteins such as the GluR2 Ca(2)-impermeable glutamate receptor subunit, superoxide dismutase 2, and the GLUT2 glucose transporter were significantly decreased in AD/GAL- nucleus basalis neurons compared to NCI and AD/GAL+ neurons. By contrast, mRNAs encoding calpain catalytic and regulatory subunits, which may contribute to cell death in AD, were increased in AD/GAL- compared to NCI and AD/GAL+ neurons. Hence, GAL fiber hyperinnervation appears to preserve the expression of genes subserving multiple neuroprotective pathways suggesting that GAL overexpression regulates CBF neuron survival in AD
— id: 133749, year: 2009, vol: 18, page: 885, stat: Journal Article,

Cortical alpha 7 nicotinic acetylcholine receptor and beta -amyloid levels in early Alzheimer disease
Ikonomovic, Milos D; Wecker, Lynn; Abrahamson, Eric E; Wuu, Joanne; Counts, Scott E; Ginsberg, Stephen D; Mufson, Elliott J; DeKosky, Steven T
2009 ;66(5):646-651, Archives of neurology
Objective: To examine alpha 7 nicotinic acetylcholine receptor (nAChR) binding and (3-amyloid (Abeta ) peptide load in superior frontal cortex (SFC) across clinical and neuropathological stages of Alzheimer disease (AD). Design: Quantitative measures of alpha 7 nAChR by [superscript 3H]methyllycaconitine binding and Abeta concentration by enzyme-linked immunosorbent assay in SFC were compared across subjects with antemortem clinical classification of no cognitive impairment, mild cognitive impairment, or mild to moderate AD, and with postmortem neuropathological diagnoses. Setting: Academic medical center. Subjects: Twenty-nine elderly retired clergy. Main Outcome Measures: Quantitative measures of alpha 7 nAChR binding and Abeta peptide concentration in SFC. Results: Higher concentrations of total Abeta peptide in SFC were associated with clinical diagnosis of mild to moderate AD (P = .02), lower Mini-Mental State Examination scores (P = .003), presence of cortical Ap plaques (P = .02), and likelihood of AD diagnosis by the National Institute on Aging-Reagan criteria (P = .002). Increased alpha 7 nAChR binding was associated with National Institute on Aging-Reagan diagnosis (P = .02) and, albeit weakly, the presence of cortical Abeta plaques (P = .08). There was no correlation between the 2 biochemical measures. Conclusions: These observations suggest that during the clinical progression from normal cognition to neurodegenerative disease state, total Abeta peptide concentration increases while alpha 7 nAChRs remain relatively stable in SFC. Regardless of subjects' clinical status, however, elevated alpha 7 nAChR binding is associated with increased Abeta plaque pathology, supporting the hypothesis that cellular expression of these receptors may be upregulated selectively in Abeta plaque-burdened brain areas.
— id: 106218, year: 2009, vol: 66, page: 646, stat: Journal Article,

Systemic pathology in aged mouse models of Down's syndrome and Alzheimer's disease
Levine, Seymour; Saltzman, Arthur; Levy, Efrat; Ginsberg, Stephen D
2009 Feb;86(1):18-22, Experimental & molecular pathology
Down's syndrome (DS) in humans is caused by trisomy of chromosome 21 (HSA 21). DS patients have a variety of pathologies, including mental retardation and an unusually high incidence of leukemia or lymphoma such as megakaryocytic leukemia. Individuals with DS develop the characteristic neuropathological hallmarks of Alzheimer's disease (AD) in early adulthood, generally by the fourth decade of life. There are several mouse models of DS that have a segmental trisomy of mouse chromosome 16 (MMU 16) with triplicated genes orthologous to HSA 21. These mice display neurodegeneration similar to DS. Although brain pathology in DS models is known, little information is available about other organs. We studied the extraneural pathology in aged DS mice (Ts65Dn, Ts2 and Ts1Cje aged 8 to 24 months) as well as other mouse models of neurodegeneration, including presenilin (PS), amyloid-beta precursor protein (APP), and tau (hTau and JNPL) transgenic mice. An increased incidence of peripheral amyloidosis, positive for amyloid A (AA) but not amyloid-beta peptide (A beta), was found in APP over-expressing and tauopathic mice as compared to non-transgenic (ntg) littermates or to DS mouse models. A higher incidence of lymphoma was found in the DS models, including Ts1Cje that is trisomic for a small segment of MMU 16 not including the App gene, but not in the APP over-expressing mice, suggesting that high APP expression is not the cause of lymphoma in DS. The occurrence of lymphomas in mouse DS models is of interest in relation to the increased incidence of malignant conditions in human DS
— id: 95847, year: 2009, vol: 86, page: 18, stat: Journal Article,

Decreased brain-derived neurotrophic factor depends on amyloid aggregation state in transgenic mouse models of Alzheimer's disease
Peng, Shiyong; Garzon, Diego J; Marchese, Monica; Klein, William; Ginsberg, Stephen D; Francis, Beverly M; Mount, Howard T J; Mufson, Elliott J; Salehi, Ahmad; Fahnestock, Margaret
2009 Jul 22;29(29):9321-9329, Journal of neuroscience
Downregulation of brain-derived neurotrophic factor (BDNF) in the cortex occurs early in the progression of Alzheimer's disease (AD). Since BDNF plays a critical role in neuronal survival, synaptic plasticity, and memory, BDNF reduction may contribute to synaptic and cellular loss and memory deficits characteristic of AD. In vitro evidence suggests that amyloid-beta (A beta) contributes to BDNF downregulation in AD, but the specific A beta aggregation state responsible for this downregulation in vivo is unknown. In the present study, we examined cortical levels of BDNF mRNA in three different transgenic AD mouse models harboring mutations in APP resulting in A beta overproduction, and in a genetic mouse model of Down syndrome. Two of the three A beta transgenic strains (APP(NLh) and TgCRND8) exhibited significantly decreased cortical BDNF mRNA levels compared with wild-type mice, whereas neither the other strain (APP(swe)/PS-1) nor the Down syndrome mouse model (Ts65Dn) was affected. Only APP(NLh) and TgCRND8 mice expressed high A beta(42)/A beta(40) ratios and larger SDS-stable A beta oligomers (approximately 115 kDa). TgCRND8 mice exhibited downregulation of BDNF transcripts III and IV; transcript IV is also downregulated in AD. Furthermore, in all transgenic mouse strains, there was a correlation between levels of large oligomers, A beta(42)/A beta(40), and severity of BDNF decrease. These data show that the amount and species of A beta vary among transgenic mouse models of AD and are negatively correlated with BDNF levels. These findings also suggest that the effect of A beta on decreased BDNF expression is specific to the aggregation state of A beta and is dependent on large oligomers
— id: 135249, year: 2009, vol: 29, page: 9321, stat: Journal Article,

Terminal Continuation (TC) RNA Amplification Enables Expression Profiling Using Minute RNA Input Obtained from Mouse Brain
Alldred, Melissa J; Che, Shaoli; Ginsberg, Stephen D
2008 Nov;9(11):2091-2104, International journal of molecular sciences
A novel methodology named terminal continuation (TC) RNA amplification has been developed to amplify RNA from minute amounts of starting material. Utility of the TC RNA amplification method is demonstrated with two new modifications including obviating the need for second strand synthesis, and purifying the amplification template using column filtration prior to in vitro transcription (IVT). Using four low concentrations of RNA extracted from mouse brain (1, 10, 25 and 50 ng), one round TC RNA amplification was compared to one round amplified antisense RNA (aRNA) in conjunction with column filtration and drop dialysis purification. The TC RNA amplification without second strand synthesis performed extremely well on custom-designed cDNA array platforms, and column filtration was found to provide higher positive detection of individual clones when hybridization signal intensity was subtracted from corresponding negative control hybridization signal levels. Results indicate that TC RNA amplification without second strand synthesis, in conjunction with column filtration, is an excellent method for RNA amplification from extremely small amounts of input RNA from mouse brain and postmortem human brain, and is compatible with microaspiration strategies and subsequent microarray analysis
— id: 101275, year: 2008, vol: 9, page: 2091, stat: Journal Article,

Galanin hyperinnervation upregulates choline acetyltransferase expression in cholinergic basal forebrain neurons in Alzheimer's disease
Counts, Scott E; He, Bin; Che, Shaoli; Ginsberg, Stephen D; Mufson, Elliott J
2008 ;5(3-4):228-231, Neuro-degenerative diseases
BACKGROUND: Fibers containing galanin (GAL) enlarge and hyperinnervate cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons in late-stage Alzheimer's disease (AD), yet the physiological consequences of this phenomenon are unclear. OBJECTIVE: To determine whether GAL hyperinnervation of cholinergic NB neurons modulates the expression of genes critical to cholinergic transmission [e.g. acetylcholine (ACh) metabolism and ACh receptors] in AD. METHODS: Single-cell gene expression profiling was used to compare cholinergic mRNA levels in non-GAL-hyperinnervated NB neurons in tissue autopsied from cases classified as having no cognitive impairment (NCI) or late-stage AD (AD/GAL-) and in GAL-hyperinnervated (AD/GAL+) NB neurons from the same AD subjects. RESULTS: AD/GAL+ cells displayed a significant upregulation in choline acetyltransferase (ChAT) mRNA expression compared to NCI and AD/GAL- cells. CONCLUSION: GAL fiber hyperinnervation of cholinergic NB neurons upregulates the expression of ChAT, the synthetic enzyme for ACh, suggesting that GAL regulates the cholinergic tone of CBF neurons in AD
— id: 135335, year: 2008, vol: 5, page: 228, stat: Journal Article,

Transcriptional profiling of small samples in the central nervous system
Ginsberg, Stephen D
2008 ;439:147-158, Methods in molecular biology
RNA amplification is a series of molecular manipulations designed to amplify genetic signals from small quantities of starting materials (including single cells and homogeneous populations of individual cell types) for microarray analysis and other downstream genetic methodologies. A novel methodology named terminal continuation (TC) RNA amplification has been developed in this laboratory to amplify RNA from minute amounts of starting material. Briefly, an RNA synthesis promoter is attached to the 3' and/or 5' region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications, including gene expression profiling, microarray analysis, and cDNA library/subtraction library construction. Input sources of RNA can originate from a myriad of in vivo and in vitro tissue sources. Moreover, a variety of fixations can be employed, and tissues can be processed for histochemistry or immunocytochemistry prior to microdissection for TC RNA amplification, allowing for tremendous cell type and tissue specificity of downstream genetic applications
— id: 78372, year: 2008, vol: 439, page: 147, stat: Journal Article,

Dysregulation of brain APP in the Ts65Dn Down syndrome mouse
Choi, JH; Mazzella, MJ; Berger, JD; Cataldo, AM; Ginsberg, SD; Levy, E; Nixon, RA; Mathews, PM
2007 AUG ;102(3):131-131, Journal of neurochemistry
— id: 74183, year: 2007, vol: 102, page: 131, stat: Journal Article,

alpha 7 nicotinic receptor up-regulation in cholinergic basal forebrain neurons in Alzheimer disease
Counts, SE; He, B; Che, SL; Ikonomovic, MD; DeKosky, ST; Ginsberg, SD; Mufson, EJ
2007 DEC ;64(12):1771-1776, Archives of neurology
Background: Dysfunction of basocortical cholinergic projection neurons of the nucleus basalis (NB) correlates with cognitive deficits in Alzheimer disease (AD). Nucleus basalis neurons receive cholinergic inputs and express nicotinic acetylcholine receptors (nAChRs) and muscarinic AChRs (mAChRs), which may regulate NB neuron activity in AD. Although alterations in these AChRs occur in the AD cortex, there is little information detailing whether defects in nAChR and mAChR gene expression occur in cholinergic NB neurons during disease progression. Objective: To determine whether nAChR and mAChR gene expression is altered in cholinergic NB neurons during the progression of AD. Design: Individual NB neurons from subjects diagnosed ante mortem as having no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild to moderate AD were analyzed by single-cell AChR expression profiling via custom-designed microarrays. Setting: Academic research. Participants: Participants were members of the Rush Religious Orders Study cohort. Main Outcome Measures: Real-time quantitative polymerase chain reaction was performed to validate micro-array findings. Results: Cholinergic NB neurons displayed a statistically significant up-regulation of alpha 7 nAChR messenger RNA expression in subjects with mild to moderate AD compared with those with NCI and MCI (P<.001). No differences were found for other nAChR and mAChR sub-types across the cohort. Expression levels of alpha 7 nAChRs were inversely associated with Global Cognitive Score and with Mini-Mental State Examination performance. Conclusions: Up-regulation of alpha 7 nAChRs may signal a compensatory response to maintain basocortical cholinergic activity during AD progression. Alternatively, putative competitive interactions of this receptor with beta-amyloid may provide a pathogenic mechanism for NB dysfunction. Increasing NB alpha 7 nAChR expression may serve as a marker for the progression of AD
— id: 75422, year: 2007, vol: 64, page: 1771, stat: Journal Article,

Cholinotrophic molecular substrates of mild cognitive impairment in the elderly
Mufson, EJ; Counts, SE; Fahnestock, M; Ginsberg, SD
2007 SEP ;4(4):340-350, Current Alzheimer research
Cholinergic nucleus basalis (NB) neurons provide the major cholinergic innervation to the cortical mantle, are selectively vulnerable in late stage Alzheimer's disease (AD) and require the neurotrophin, nerve growth factor (NGF) and its receptors (TrkA and p75(NTR)), for their survival. The molecular events underlying the demise of these neurons in AD were investigated using tissue harvested from participants in a longitudinal clinical pathological study of aging and AD who agreed to an annual clinical evaluation providing a categorization of no cognitive impairment (NCI), mild cognitive impairment (MCI) or AD and postmortem brain donation. Although the number of choline acetyltransferase (ChAT)positive neurons was unchanged, TrkA and p75NTR receptor-containing neurons, which co-localize with ChAT, were significantly reduced in the NB of subjects with MCI and AD compared to those with NCI. These observations indicate a phenotypic down-regulation rather than frank NB neuronal degeneration in MCI. Expression profiling of single cholinergic NB neurons revealed TrkA but not p75NTR mRNA is reduced in MCI, suggesting that decreased neurotrophin responsiveness may be an early biomarker for AD. The NGF precursor molecule, proNGF, is increased in the cortex in MCI and AD. Since proNGF accumulates in the presence of reduced cortical TrkA and sustained levels of p75NTR, a shift in the balance between cell survival and death molecules may occur in prodromal AD. Coincident with these phenomena, brain derived neurotrophic factor (BDNF) and its precursor molecule, proBDNF, are reduced in the MCI cortex, potentially depriving CBF neurons of additional trophic factor support. Moreover, there is. a shift in the ratio of 3 repeat tau to 4 repeat tau gene expression, whereas total tau message is stable in NB neurons during the disease process. These data suggest there is a shift in cholinotrophic molecular events in MCI and early AD which may lead to cell dysfunction and eventual cell death over the course of the disease. These findings support the concept that from a neurotrophic pathobiologic perspective, MCI is already early AD
— id: 74689, year: 2007, vol: 4, page: 340, stat: Journal Article,

Galanin fiber hypertrophy within the cholinergic nucleus basalis during the progression of Alzheimer's disease
Counts, SE; Chen, EY; Che, SL; Ikonomovic, MD; Wuu, J; Ginsberg, SD; DeKosky, ST; Mufson, EJ
2006 FEB ;21(4):205-214, Dementia geriatric & cognitive disorders
Galanin (GAL)-containing fibers enlarge and hyperinnervate remaining cholinergic basal forebrain (CBF) neurons within the anterior nucleus basalis ( NB) in late-stage Alzheimer's disease (AD). Whether GAL hypertrophy occurs in the CBF in the prodromal or early stages of AD remains unknown. The present study used GAL immunohistochemistry and an unbiased semiquantitative scoring method to evaluate GAL innervation in the anterior NB of subjects clinically diagnosed as having no cognitive impairment, mild cognitive impairment or early-stage (mild/moderate) AD. There was no difference in GAL fiber staining within the anterior NB across the three clinical groups examined. Furthermore, GAL fiber innervation was not correlated with the number of NB neurons expressing the nerve growth factor receptors p75(NTR) or TrkA or with cortical choline acetyltransferase activity in the same cases. Single-cell gene expression analysis demonstrated that cholinergic NB neurons express mRNA for the GAL receptors GALR1, GALR2 and GALR3, yet the levels of these mRNAs were unchanged across the three diagnostic groups. These observations indicate that GAL hypertrophy within the anterior NB subfield is a late-stage AD response, which may play a role in regulating the cholinergic tone of remaining basocortical projection neurons
— id: 62907, year: 2006, vol: 21, page: 205, stat: Journal Article,

Shift in the ratio of three-repeat tau and four-repeat tau mRNAs in individual cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease
Ginsberg, SD; Che, S; Counts, SE; Mufson, EJ
2006 MAR ;96(5):1401-1408, Journal of neurochemistry
Molecular mechanisms underlying tauopathy remain undetermined. In the current study, single cell gene expression profiling was coupled with custom-designed cDNA array analysis to evaluate tau expression and other cytoskeletal elements within individual neuronal populations in patients with no cognitive impairment (NCI), mild cognitive impairment (MCI), and Alzheimer's disease (AD). Results revealed a shift in the ratio of three-repeat tau (3Rtau) to four-repeat tau (4Rtau) mRNAs within individual human cholinergic basal forebrain (CBF) neurons within nucleus basalis (NB) and CA1 hippocampal neurons during the progression of AD, but not during normal aging. A shift in 3Rtau to 4Rtau may precipitate a cascade of events in the selective vulnerability of neurons, ultimately leading to frank neurofibrillary tangle (NFT) formation in tauopathies including AD
— id: 62633, year: 2006, vol: 96, page: 1401, stat: Journal Article,

Single cell gene expression profiling in Alzheimer's disease
Ginsberg, Stephen D; Che, Shaoli; Counts, Scott E; Mufson, Elliott J
2006 Jul;3(3):302-318, NeuroRx
Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD
— id: 140351, year: 2006, vol: 3, page: 302, stat: Journal Article,

Down regulation of trk but not p75NTR gene expression in single cholinergic basal forebrain neurons mark the progression of Alzheimer's disease
Ginsberg, Stephen D; Che, Shaoli; Wuu, Joanne; Counts, Scott E; Mufson, Elliott J
2006 Apr;97(2):475-487, Journal of neurochemistry
Dysfunction of cholinergic basal forebrain (CBF) neurons of the nucleus basalis (NB) is a cardinal feature of Alzheimer's disease (AD) and correlates with cognitive decline. Survival of CBF neurons depends upon binding of nerve growth factor (NGF) with high-affinity (trkA) and low-affinity (p75(NTR)) neurotrophin receptors produced within CBF neurons. Since trkA and p75(NTR) protein levels are reduced within CBF neurons of people with mild cognitive impairment (MCI) and mild AD, trkA and/or p75(NTR) gene expression deficits may drive NB degeneration. Using single cell expression profiling methods coupled with custom-designed cDNA arrays and validation with real-time quantitative PCR (qPCR) and in situ hybridization, individual cholinergic NB neurons displayed a significant down regulation of trkA, trkB, and trkC expression during the progression of AD. An intermediate reduction was observed in MCI, with the greatest decrement in mild to moderate AD as compared to controls. Importantly, trk down regulation is associated with cognitive decline measured by the Global Cognitive Score (GCS) and the Mini-Mental State Examination (MMSE). In contrast, there is a lack of regulation of p75(NTR) expression. Thus, trk defects may be a molecular marker for the transition from no cognitive impairment (NCI) to MCI, and from MCI to frank AD
— id: 64458, year: 2006, vol: 97, page: 475, stat: Journal Article,

Functional genomic methodologies
Ginsberg, Stephen D; Mirnics, Karoly
2006 ;158:15-40, Progress in brain research
The ability to form tenable hypotheses regarding the neurobiological basis of normative functions as well as mechanisms underlying neurodegenerative and neuropsychiatric disorders is often limited by the highly complex brain circuitry and the cellular and molecular mosaics therein. The brain is an intricate structure with heterogeneous neuronal and nonneuronal cell populations dispersed throughout the central nervous system. Varied and diverse brain functions are mediated through gene expression, and ultimately protein expression, within these cell types and interconnected circuits. Large-scale high-throughput analysis of gene expression in brain regions and individual cell populations using modern functional genomics technologies has enabled the simultaneous quantitative assessment of dozens to hundreds to thousands of genes. Technical and experimental advances in the accession of tissues, RNA amplification technologies, and the refinement of downstream genetic methodologies including microarray analysis and real-time quantitative PCR have generated a wellspring of informative studies pertinent to understanding brain structure and function. In this review, we outline the advantages as well as some of the potential challenges of applying high throughput functional genomics technologies toward a better understanding of brain tissues and diseases using animal models as well as human postmortem tissues
— id: 70305, year: 2006, vol: 158, page: 15, stat: Journal Article,

Characterization of the putative cholesterol transport protein metastatic lymph node 64 in the brain
King, SR; Smith, AGA; Alpy, F; Tomasetto, C; Ginsberg, SD; Lamb, DJ
2006 JUN ;139(3):1031-1038, Neuroscience
Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport. (c) 2006 IBRO. Published by Elsevier Ltd. All rights reserved
— id: 64393, year: 2006, vol: 139, page: 1031, stat: Journal Article,

Neuronal gene expression profiling: uncovering the molecular biology of neurodegenerative disease
Mufson, EJ; Counts, SE; Che, SL; Ginsberg, SD
2006 MAY ;158(5):197-222, Progress in brain research
The development of gene array techniques to quantify expression levels of dozens to thousands of genes simultaneously within selected tissue samples from control and diseased brain has enabled researchers to generate expression profiles of vulnerable neuronal populations in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, and Creutzfeld Jakob disease. Intriguingly, gene expression analysis reveals that vulnerable brain regions in many of these diseases share putative pathogenetic alterations in common classes of genes, including decrements in synaptic transcript levels and increments in immune response transcripts. Thus, gene expression profiles of diseased neuronal populations may reveal mechanistic clues to the molecular pathogenesis underlying various neurological diseases and aid in identifying potential therapeutic targets. This chapter will review how regional and single cell gene array technologies have advanced our understanding of the genetics of human neurological disease
— id: 72997, year: 2006, vol: 158, page: 197, stat: Journal Article,

Shifting sands of cholinergic neurotrophic activity in prodromal Alzheimer disease
Mufson, EJ; Counts, SE; Ginsberg, SD
2006 APR ;27(5):S15-S15, Neurobiology of aging
— id: 62895, year: 2006, vol: 27, page: S15, stat: Journal Article,

Endosome dysfunction in Alzheimer's disease: Genetic links and implications for synapse failure and neurodegeneration
Nixon, RA; Cataldo, A; Mathews, P; Jiang, Y; Ginsberg, SD; Peterhoff, C
2006 DEC ;31(2):S28-S28, Neuropsychopharmacology
— id: 70911, year: 2006, vol: 31, page: S28, stat: Journal Article,

RNA amplification of bromodeoxyuridine labeled newborn neurons in the monkey hippocampus
Counts, Scott E; Chen, Er-Yun; Ginsberg, Stephen D; Kordower, Jeffrey H; Mufson, Elliott J
2005 Jun 15;144(2):197-201, Journal of neuroscience methods
Neurogenesis has been demonstrated in the adult mammalian hippocampus by the immunohistochemical identification of cells co-labeled with the neuronal marker NeuN and bromodeoxyuridine (BrdU), a marker for DNA synthesis. Whether these newly born neurons exhibit a genetic signature similar to that of existing hippocampal cells remains unknown. Recent advances in single cell RNA amplification techniques provide a unique method for profiling the mRNA complement of cells developed during adult neurogenesis. Standard protocols for identifying BrdU-positive cells requires an acid denaturation step that may preclude the amplification of cellular RNA for expression analysis. We first tested whether the BrdU reaction product was visible in monkey hippocampal tissue following treatment with dilutions of HCl (2-0.2 M) or citric acid (1.0-0.1 M). BrdU-labeled cells were visible only in tissue sections treated with 2 M HCl. RNA amplification was not compromised in cells dual-labeled for BrdU and NeuN using the 2 M HCl acid denaturation step. These cells express mRNAs encoding a wide variety of functional protein subclasses including glutamate receptors. The present study demonstrates for the first time that BrdU immunohistochemisty is compatable with gene array technology in the primate hippocampus to evaluate subclasses of genes in newborn neurons
— id: 60999, year: 2005, vol: 144, page: 197, stat: Journal Article,

Expression profiling in the aging brain: a perspective
Galvin, James E; Ginsberg, Stephen D
2005 Nov;4(4):529-547, Ageing research reviews
To evaluate molecular events associated with the aging process in animal models and human tissues, microarray analysis is performed at the regional and cellular levels to define transcriptional patterns or mosaics that may lead to better understanding of the mechanism(s) that drive senescence. In this review, we outline the experimental and analytical issues associated with high-throughput genomic analyses in aging brain and other tissues for a comprehensive evaluation of the current state of microarray analysis in aging paradigms. Ultimately, the goal of these studies is to apply functional genomics and proteomics approaches to aging research to develop new tools to assess age in cell- and tissue-specific manners in order to develop aging biomarkers for pharmacotherapeutic interventions and disease prevention
— id: 110039, year: 2005, vol: 4, page: 529, stat: Journal Article,

Glutamatergic neurotransmission expression profiling in the mouse hippocampus after perforant-path transection
Ginsberg, Stephen D
2005 Dec;13(12):1052-1061, American journal of geriatric psychiatry
OBJECTIVE: The goal is to determine cellular and molecular mechanisms that regulate regenerative and neurodegenerative responses within the adult mouse dentate gyrus after axotomy of the principal glutamatergic input, the perforant path (PP). METHODS: A 'molecular fingerprint' of the dentate gyrus was generated to provide an extensive, concurrent representation of genes, with an emphasis on glutamate receptor subunits and related markers of glutamatergic neurotransmission. Reorganization of the hippocampal formation was evaluated by regional microdissection of the dentate gyrus, followed by terminal continuation RNA amplification and custom-designed cDNA microarray analysis after unilateral PP transections at five time-points (0, 1, 10, 14, and 30 days post-lesion). Gene-expression profiles garnered from the ipsilateral side of PP transected hippocampal formation (including the dentate gyrus) were compared and contrasted with those of naive subjects, sham surgical subjects, and mice subjected to unilateral occipital cortex lesions. Specific gene array observations were validated by immunoblot analysis. RESULTS: Results indicated selective regulation of specific transcripts, including AMPA and NMDA glutamate-receptor subunits, excitatory amino acid transporters, glutamate receptor interacting protein genes, and glial-associated markers across the time-course of the lesion study. CONCLUSION: The goal was to identify messenger RNAs from specific classes of relevant transcripts that change over time in relationship to the synaptic and cellular alterations to help understand mechanisms that underlie lesion-induced synaptic plasticity
— id: 60249, year: 2005, vol: 13, page: 1052, stat: Journal Article,

RNA amplification strategies for small sample populations
Ginsberg, Stephen D
2005 Nov;37(3):229-237, Methods
Advances in high throughput cloning strategies have led to sequencing of the human genome as well as progress in the sequencing of the genome of several other species. Consequently, the field of molecular genetics is blossoming into a multidisciplinary entity that is revolutionizing the way researchers evaluate a myriad of critical concepts such as development, homeostasis, and disease pathogenesis. There is tremendous interest in the quantitative assessment of tissue-specific expression of both newly identified and well characterized specific genes and proteins. At present, an ideal approach is to assess gene expression in single elements recorded physiologically in living preparations or by immunocytochemical or histochemical methods in fixed cells in vitro or in vivo. The quantity of RNA harvested from individual cells is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction based analyses, and linear RNA amplification including amplified antisense RNA amplification and a newly developed terminal continuation RNA amplification methodology have been developed for use in combination with microdissection procedures and cDNA/oligonucleotide microarray platforms. RNA amplification is a series of intricate procedures to amplify genetic signals from minute quantities of starting materials for microarray analysis and other downstream genetic methodologies. RNA amplification procedures effectively generate quantities of RNA through in vitro transcription. The present report illustrates practical usage of RNA amplification technologies within the context of regional, population cell, and single cell analyses in the brain
— id: 60250, year: 2005, vol: 37, page: 229, stat: Journal Article,

Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons
Ginsberg, Stephen D; Che, Shaoli
2005 Jun 20;487(1):107-118, Journal of comparative neurology
The hippocampus contains several distinct cell types that are interconnected by a well-characterized series of synaptic circuits. To evaluate molecular and cellular signatures of individual cell types within the normal adult human hippocampal formation, expression profile analysis was performed on individual CA1 and CA3 pyramidal neurons using a novel single cell RNA amplification methodology coupled with custom-designed cDNA array analysis. Populations of CA1 and CA3 neurons were also compared with regional dissections of the hippocampus from the same tissue sections. Molecular fingerprint comparison of cresyl violet-stained CA1 and CA3 pyramidal neurons microaspirated from the hippocampus of normal control subjects indicated significant differences in relative expression levels for approximately 16% (20 of 125) genes evaluated on the custom-designed cDNA array platform. Significant differences were observed for several transcripts relevant to the structure and function of hippocampal neurons, including specific glutamate receptors, gamma-aminobutyric acid (GABA) A receptors, cytoskeletal elements, dopamine receptors, and immediate-early genes. Compared with the regional assessment of gene expression, both CA1 and CA3 neurons displayed a relative enrichment of classes of transcripts that included glutamate receptors, transporters, and interacting proteins, GABA receptors and transporters, synaptic-related markers, and catecholamine receptors and transporters. In contrast, the regional hippocampal dissection had an increased level of gene expression for cytoskeletal elements as well as glial-associated markers. Expression profile analysis illustrates the importance of evaluating individual cellular populations within a functional circuit and may help define elements that confer unique properties to individual populations of hippocampal neurons under normal and diseased conditions
— id: 56000, year: 2005, vol: 487, page: 107, stat: Journal Article,

Expression profiling using human tissues in combination with RNA amplification and microarray analysis: assessment of Langerhans cell histiocytosis
McClain, KL; Cai, YH; Hicks, J; Peterson, LE; Yan, XT; Che, S; Ginsberg, SD
2005 MAY ;28(3):279-290, Amino acids
Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses
— id: 55700, year: 2005, vol: 28, page: 279, stat: Journal Article,

Amplification of RNA transcripts using terminal continuation
Che, Shaoli; Ginsberg, Stephen D
2004 Jan;84(1):131-137, Laboratory investigation
A new methodology has been developed to amplify RNA from minute amounts of starting material. Specifically, an efficient means of second-strand (ss) cDNA synthesis using a sequence-specific 'terminal continuation' (TC) method is demonstrated. An RNA synthesis promoter is attached to the 3' and/or 5' region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications including gene expression profiling, cDNA microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translations and downstream proteomic applications. The TC RNA amplification methodology offers high sensitivity, flexibility, and throughput capabilities. A likely mechanism is that the TC primer binds preferentially to GC-rich CpG islands flanking 5' regions of DNA that contain promoter sequences. Following TC RNA amplification, a large proportion of genes can be assessed quantitatively as evidenced by bioanalysis and cDNA microarray analysis in mouse and human postmortem brain tissues
— id: 42641, year: 2004, vol: 84, page: 131, stat: Journal Article,

Reduction of cortical TrkA but not p75(NTR) protein in early-stage Alzheimer's disease
Counts, Scott E; Nadeem, Muhammad; Wuu, Joanne; Ginsberg, Stephen D; Saragovi, H Uri; Mufson, Elliott J
2004 Oct;56(4):520-531, Annals of neurology
Degeneration of cholinergic nucleus basalis (NB) cortical projection neurons is associated with cognitive decline in late-stage Alzheimer's disease (AD). NB neuron survival is dependent on coexpression of the nerve growth factor (NGF) receptors p75(NTR) and TrkA, which bind NGF in cortical projection sites. We have shown previously a significant reduction of NB perikarya expressing p75(NTR) and TrkA protein during the early stages of AD. Whether there is a concomitant reduction in cortical levels of these receptors during the progression of AD is unknown. p75(NTR) and TrkA protein was evaluated by quantitative immunoblotting in five cortical regions (anterior cingulate, superior frontal, superior temporal, inferior parietal, and visual cortex) of individuals clinically diagnosed with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD, or severe AD. Cortical p75(NTR) levels were stable across the diagnostic groups. In contrast, TrkA levels were reduced approximately 50% in mild/moderate and severe AD compared with NCI and MCI in all regions except visual cortex. Mini-Mental Status Examination scores correlated with TrkA levels in anterior cingulate, superior frontal, and superior temporal cortex. The selective reduction of cortical TrkA levels relative to p75(NTR) may have important consequences for cholinergic NB function during the transition from MCI to AD
— id: 61000, year: 2004, vol: 56, page: 520, stat: Journal Article,

Expression profiling and pharmacotherapeutic development in the central nervous system
Galvin, James E; Ginsberg, Stephen D
2004 Oct-Dec;18(4):264-269, Alzheimer disease & associated disorders
Expression profiling data is available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as cellular development, differentiation, normative function, and disease pathogenesis. The central nervous system is an ideal structure to evaluate relationships between functional genomics and expression data. Recent developments in gene array technologies, specifically cDNA microarray platforms, have made it easier to try to understand the multiplicity of gene alterations that occur within the brains of animal models and postmortem human tissues. However, unlike structures have one principal cell type, the brain contains diverse populations of phenotypically distinct cell types. A goal of modern molecular and cellular neuroscience is to assay gene expression from homogeneous populations of cells within a defined region without potential contamination by expression profiles of adjacent neuronal subtypes and non-neuronal cells. This is a difficult task that demands a multidisciplinary approach that is highlighted in this review within the context of neurodegenerative pathology
— id: 110169, year: 2004, vol: 18, page: 264, stat: Journal Article,

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus
Ginsberg, Stephen D; Che, Shaoli
2004 Aug;84(8):952-962, Laboratory investigation
The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies
— id: 44700, year: 2004, vol: 84, page: 952, stat: Journal Article,

Single-cell gene expression analysis: implications for neurodegenerative and neuropsychiatric disorders
Ginsberg, Stephen D; Elarova, Irina; Ruben, Marc; Tan, Fengzhu; Counts, Scott E; Eberwine, James H; Trojanowski, John Q; Hemby, Scott E; Mufson, Elliott J; Che, Shaoli
2004 Jun;29(6):1053-1064, Neurochemical research
Technical and experimental advances in microaspiration techniques, RNA amplification, quantitative real-time polymerase chain reaction (qPCR), and cDNA microarray analysis have led to an increase in the number of studies of single-cell gene expression. In particular, the central nervous system (CNS) is an ideal structure to apply single-cell gene expression paradigms. Unlike an organ that is composed of one principal cell type, the brain contains a constellation of neuronal and noneuronal populations of cells. A goal is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and noneuronal cells. The unprecedented resolution afforded by single-cell RNA analysis in combination with cDNA microarrays and qPCR-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease states. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models as well as postmortem human brain tissues. This focused review illustrates the potential power of single-cell gene expression studies within the CNS in relation to neurodegenerative and neuropsychiatric disorders such as Alzheimer's disease (AD) and schizophrenia, respectively
— id: 44699, year: 2004, vol: 29, page: 1053, stat: Journal Article,

The steroidogenic acute regulatory protein is expressed in steroidogenic cells of the day-old brain
King, Steven R; Ginsberg, Stephen D; Ishii, Tomohiro; Smith, Roy G; Parker, Keith L; Lamb, Dolores J
2004 Oct;145(10):4775-4780, Endocrinology
Although recent research has focused on the fundamental role(s) of steroids synthesized de novo in the brain on development, the mechanism by which production of these neurosteroids is regulated remains unclear. Steroid production in peripheral tissues is acutely regulated by the steroidogenic acute regulatory (StAR) protein, which mediates the rate-limiting step in steroid biosynthesis: the intramitochondrial delivery of cholesterol to cytochrome P450scc for conversion to steroid. We recently demonstrated that StAR is present in discrete cell types in the adult brain, suggesting that neurosteroid production is mediated by StAR. Nevertheless, little is known regarding the presence of StAR in the developing brain. In the present study, the presence of StAR and for the first time, its homolog, the putative cholesterol transport protein metastatic lymph node 64 (MLN64), were defined in the neonatal mouse brain using immunocytochemical techniques. Both StAR and MLN64 were found to be present in the brain with staining patterns characteristic to each protein, indicating the authenticity of StAR and MLN64 immunoreactivity. Furthermore, we found MLN64 to be expressed in the adult brain as well, apparently at higher levels than StAR. Importantly, StAR protein is present in cells that also express P450scc. These data suggest that, as with the adult, neurosteroid production during development occurs through a StAR-mediated pathway
— id: 48170, year: 2004, vol: 145, page: 4775, stat: Journal Article,

Galanin in Alzheimer disease
Counts, Scott E; Perez, Sylvia E; Ginsberg, Stephen D; De Lacalle, Sonsoles; Mufson, Elliott J
2003 May;3(3):137-156, Molecular interventions
Galanin (GAL) and GAL receptors (GALR) are overexpressed in limbic brain regions associated with cognition in Alzheimer disease (AD). The functional consequences of this overexpression are unclear. Because GAL inhibits cholinergic transmission and restricts long-term potentiation in the hippocampus, GAL overexpression may exacerbate clinical features of AD. In contrast, GAL expression increases in response to neuronal injury, and galaninergic hyperinnervation prevents the decreased production of protein phosphatase 1 subtype mRNAs in cholinergic basal forebrain neurons in AD. Thus, GAL may also be neuroprotective for AD. Further elucidation of GAL activity in selectively vulnerable brain regions will help gauge the therapeutic potential of GALR ligands for the treatment of AD
— id: 61001, year: 2003, vol: 3, page: 137, stat: Journal Article,

Neuron-specific age-related decreases in dopamine receptor subtype mRNAs
Hemby, SE; Trojanowski, JQ; Ginsberg, SD
2003 FEB 3 ;456(2):176-183, Journal of comparative neurology
Age-related decline in dopamine receptor levels has been observed in regional studies of animal and human brains; however, identifying specific cellular substrates and/or alterations in distinct neuronal populations remains elusive. To evaluate whether age-related decreases in dopamine receptor subtypes are associated with specific cell populations in the hippocampus and entorhinal cortex, antisense RNA amplification was combined with cDNA array analysis to examine effects of aging on D1-D5 dopamine receptor mRNA expression levels in hippocampal CA1 pyramidal neurons and entorhinal cortex layer II stellate cells from post-mortem human brains (19-92 years). In CA1 pyramidal neurons, significant age-related decline was observed for dopamine receptor mRNAs (D1-D4, P < 0.001; D5, P < 0.05) but not for the cytoskeletal elements P-actin, three- repeat (3R) tau, and four-repeat (4R) tau, In contrast, no significant changes were observed in stellate cells across the same cohort. Thus, senescence may be a factor responsible for cell-specific decrements in dopamine receptor gene expression in one population of neurons within a circuit that is critical for learning and memory. Furthermore, these results support the hypothesis that alterations in dopaminergic function may also be related to behavioral abnormalities, such as psychosis, that occur with aging. (C) 2002 Wiley-Liss, Inc
— id: 33268, year: 2003, vol: 456, page: 176, stat: Journal Article,

Gene expression in Langerhans Cell histiocytosis
McClain, KL; Cai, YH; Hicks, J; Peterson, LE; Ginsberg, SD
2003 APR ;53(4):288A-289A, Pediatric research
— id: 38565, year: 2003, vol: 53, page: 288A, stat: Journal Article,

Human cholinergic basal forebrain: chemoanatomy and neurologic dysfunction
Mufson, EJ; Ginsberg, SD; Ikonomovic, MD; DeKosky, ST
2003 DEC ;26(4):233-242, Journal of chemical neuroanatomy
The human cholinergic basal forebrain (CBF) is comprised of magnocellular hyperchromic neurons within the septal/diagonal band complex and nucleus basalis (NB) of Meynert. CBF neurons provide the major cholinergic innervation to the hippocampus, amygdala and neocortex. They play a role in cognition and attentional behaviors, and are dysfunctional in Alzheimer's disease (AD). The human CBF displays a continuum of large cells that contain various cholinergic markers, nerve growth factor (NGF) and its cognate receptors, calbindin, glutamate receptors, and the estrogen receptors, ERalpha and ERbeta. Admixed with these cholinergic neuronal phenotypes are smaller interneurons containing the m2 muscarinic acetylcholine receptor (mAChRs), NADPH-diaphorase, GABA, calcium binding proteins and several inhibitory neuropeptides including galanin (GAL), which is over expressed in AD. Studies using human autopsy material indicate an age-related dissociation of calbindin and the glutamate receptor GluR2 within CBF neurons, suggesting that these molecules act synergistically to induce excitotoxic cell death during aging, and possibly during AD. Choline acetyltrasnferease (ChAT) activity and CBF neuron number is preserved in the cholinergic basocortical system and up regulated in the septohippocampal system during prodromal as compared with end stage AD. In contrast, the number of CBF neurons containing NGF receptors is reduced early in the disease process suggesting a phenotypic silence and not a frank loss of neurons. In end stage AD, there is a selective reduction in trkA mRNA but not p75(NTR) in single CBF cells suggesting a neurotrophic defect throughout the progression of AD. These observations indicate the complexity of the chemoanatomy of the human CBF and suggest that multiple factors play different roles in its dysfunction in aging and AD. (C) 2003 Published by Elsevier B.V
— id: 42512, year: 2003, vol: 26, page: 233, stat: Journal Article,

RNA amplification in brain tissues
Ginsberg, Stephen D; Che, Shaoli
2002 Oct;27(10):981-992, Neurochemical research
Recent developments in gene array technologies, specifically cDNA microarray platforms, have made it easier to try to understand the constellation of gene alterations that occur within the CNS. Unlike an organ that is comprised of one principal cell type, the brain contains a multiplicity of both neuronal (e.g., pyramidal neurons, interneurons, and others) and noneuronal (e.g., astrocytes, microglia, oligodendrocytes, and others) populations of cells. An emerging goal of modern molecular neuroscience is to sample gene expression from similar cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subtypes and noneuronal cells. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses and linear RNA amplifications, including a newly developed terminal continuation (TC) RNA amplification methodology, have been used in combination with single cell microdissection procedures to enable the use of cDNA microarray analysis within individual populations of cells obtained from postmortem brain samples as well as the brains of animal models of neurodegeneration
— id: 60392, year: 2002, vol: 27, page: 981, stat: Journal Article,

Gene expression profile for schizophrenia - Discrete neuron transcription patterns in the entorhinal cortex
Hemby, SE; Ginsberg, SD; Brunk, B; Arnold, SE; Trojanowski, JQ; Eberwine, JH
2002 Jul;59(7):631-640, Archives of general psychiatry
Background: Several lines of evidence indicate the altered function of the temporal lobe, including the hippocampus and entorhinal cortex (EC), is associated with schizophrenia. We used single-cell gene expression technologies to assess coordinate changes in the expression of multiple genes, including neuronal signaling and synaptic-related markers in EC layer 11 stellate neurons. Methods: We used a single-neuron microdissection technique coupled with linear antisense RNA amplification and high density/candidate gene arrays to assess coordinate changes in gene expression. The expression and relative abundance of more than 18 000 messenger RNAs were assessed from EC layer 11 stellate neurons from postmortem samples of schizophrenic and age-matched control brains. Results of this initial screen were used to perform a more specific secondary messenger RNA screen for each subject. Results: Data disclosed marked differences in expression of various G-protein-coupled receptor-signaling transcripts, glutamate receptor subunits, synaptic proteins, and other transcripts. Results of secondary screening showed significant decreases in levels of G-protein subunit ialpha1, glutamate receptor 3, N-methyl-D-aspartate receptor 1, synaptophysin, and sensory nerve action potentials 23 and 25 in the stellate neurons of schizophrenic patients. We observed down-regulation of phospholemman (a phosphoprotein associated with anion channel formation) messenger RNA and protein levels in layer II/II stellate neurons in the population with schizophrenia. Conclusions: These results provide a preliminary expression profile of schizophrenia in defined neuronal populations. Understanding the coordinated involvement of multiple genes in human disease provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention
— id: 32373, year: 2002, vol: 59, page: 631, stat: Journal Article,

An essential component in steroid synthesis, the steroidogenic acute regulatory protein, is expressed in discrete regions of the brain
King, SR; Manna, PR; Ishii, T; Syapin, PJ; Ginsberg, SD; Wilson, K; Walsh, LP; Parker, KL; Stocco, DM; Smith, RG; Lamb, DJ
2002 DEC 15 ;22(24):10613-10620, Journal of neuroscience
Recent data implicate locally produced steroids, termed neurosteroids, as regulators of neuronal function. Adrenal and gonadal steroidogenesis is controlled by changes in the steroidogenic acute regulatory protein (StAR); however, little is known about the regulation of neurosteroid production. We now demonstrate unequivocally that StAR mRNA and protein are expressed within glia and neurons in discrete regions of the mouse brain, and that glial StAR expression is inducible. Consistent with a role in de novo neurosteroidogenesis, StAR colocalizes with the cholesterol side-chain cleavage enzyme P450(scc) in both mouse and human brains. These data support a role for StAR in the production of neurosteroids and identify potential sites of active de novo steroid synthesis in the brain
— id: 33271, year: 2002, vol: 22, page: 10613, stat: Journal Article,

Gene expression profiles of cholinergic nucleus basalis neurons in Alzheimer's disease
Mufson, Elliott J; Counts, Scott E; Ginsberg, Stephen D
2002 Oct;27(10):1035-1048, Neurochemical research
Cholinergic neurons of the nucleus basalis (NB) are selectively vulnerable in Alzheimer's disease (AD), yet the molecular mechanisms associated with their dysfunction remain unknown. We used single cell RNA amplification and custom array technology to examine the expression of functional classes of mRNAs found in anterior NB neurons from normal aged and AD subjects. mRNAs encoding neurotrophin receptors, synaptic proteins, protein phosphatases, and amyloid-related proteins were evaluated. We found that trkB and trkC mRNAs were selectively down-regulated in NB neurons, whereas p75NTR mRNA levels remained stable in end stage AD. TrkA mRNA was reduced by approximately 28%, but did not reach statistical significance. There was a down-regulation of synaptophysin, synaptotagmin, and protein phosphatases PP1alpha and PP1beta mRNAs in AD. In contrast, we found a selective up-regulation of cathepsin D mRNA in NB neurons in AD brain. Thus, anterior NB neurons undergo selective alterations in gene expression in AD. These results may provide clues to the molecular pathogenesis of NB neuronal degeneration during AD
— id: 61002, year: 2002, vol: 27, page: 1035, stat: Journal Article,

Autophagic cell death: Relevance to Alzheimer disease neurodegeneration
Nixon, RA; Mathews, PM; Ginsberg, SD; Duff, K; Mohan, P; Cataldo, AM; Wegiel, J; Yu, WH; Schmidt, SD; Jacobsen, SP; Peterhoff, CM; Terio, N; Keller, JN; Hassinger, L
2002 Jul-Aug;23(1):1527-, Neurobiology of aging
— id: 32429, year: 2002, vol: 23, page: 1527, stat: Journal Article,

Expression profile of transcripts in Alzheimer's disease tangle-bearing CA1 neurons
Ginsberg SD; Hemby SE; Lee VM; Eberwine JH; Trojanowski JQ
2000 Jul;48(1):77-87, Annals of neurology
The pathogenesis of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) is poorly understood, but changes in the expression of specific messenger RNAs (mRNAs) may reflect mechanisms underlying the formation of NFTs and their consequences in affected neurons. For these reasons, we compared the relative abundance of multiple mRNAs in tangle-bearing versus normal CA1 neurons aspirated from sections of AD and control brains. Amplified antisense RNA expression profiling was performed on individual isolated neurons for analysis of greater than 18,000 expressed sequence tagged complementary DNAs (cDNAs) with cDNA microarrays, and further quantitative analyses were performed by reverse Northern blot analysis on 120 selected mRNAs on custom cDNA arrays. Relative to normal CA1 neurons, those harboring NFTs in AD brains showed significant reductions in several classes of mRNAs that are known to encode proteins implicated in AD neuropathology, including phosphatases/kinases, cytoskeletal proteins, synaptic proteins, glutamate receptors, and dopamine receptors. Because cathepsin D mRNA was upregulated in NFT-bearing CA1 neurons in AD brains, we performed immunohistochemical studies that demonstrated abundant cathepsin D immunoreactivity in the same population of tangle-bearing CA1 neurons. In addition, levels of mRNAs encoding proteins not previously implicated in AD were reduced in CA1 tangle-bearing neurons, suggesting that these proteins (eg, activity-regulated cytoskeleton-associated protein, focal adhesion kinase, glutaredoxin, utrophin) may be novel mediators of NFT formation or degeneration in affected neurons. Thus, the profile of mRNAs differentially expressed by tangle-bearing CA1 neurons may represent a 'molecular fingerprint' of these neurons, and we speculate that mRNA expression profiles of diseased neurons in AD may suggest new directions for AD research or identify novel targets for developing more effective AD therapies
— id: 24714, year: 2000, vol: 48, page: 77, stat: Journal Article,

Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques
Ginsberg SD; Crino PB; Hemby SE; Weingarten JA; Lee VM; Eberwine JH; Trojanowski JQ
1999 Feb;45(2):174-181, Annals of neurology
The sequestration of RNA in Alzheimer's disease (AD) senile plaques (SPs) and the production of intraneuronal amyloid-beta peptides (Abeta) prompted analysis of the mRNA profile in single immunocytochemically identified SPs in sections of AD hippocampus. By using amplified RNA expression profiling, polymerase chain reaction, and in situ hybridization, we assessed the presence and abundance of 51 mRNAs that encode proteins implicated in the pathogenesis of AD. The mRNAs in SPs were compared with those in individual CA1 neurons and the surrounding neuropil of control subjects. The remarkable demonstration here, that neuronal mRNAs predominate in SPs, implies that these mRNAs are nonproteinaceous components of SPs, and, moreover, that mRNAs may interact with Abeta protein and that SPs form at sites where neurons degenerate in the AD brain
— id: 24716, year: 1999, vol: 45, page: 174, stat: Journal Article,

Accumulation of intracellular amyloid-beta peptide (A beta 1-40) in mucopolysaccharidosis brains
Ginsberg SD; Galvin JE; Lee VM; Rorke LB; Dickson DW; Wolfe JH; Jones MZ; Trojanowski JQ
1999 Aug;58(8):815-824, Journal of neuropathology & experimental neurology
To evaluate whether in vivo accumulations of heparan sulfate caused by inborn errors in the metabolism of glycosaminoglycans lead to the formation of neurofibrillary tangles and/or senile plaques, as seen in Alzheimer disease (AD), we studied postmortem brains from 9 patients, ages 1 to 42 years, with mucopolysaccharidosis (MPS). The brains of patients with Hurler's syndrome (MPS I: n = 5) and Sanfilippo's syndrome (MPS III; n = 4) as well as from caprine MPS IIID and murine MPS VII models were evaluated by thioflavine-S staining and by immunohistochemistry using antibodies directed against heparan sulfate proteoglycans, hyperphosphorylated tau, amyloid-beta peptide precursor proteins (APP), and amyloid-beta peptides (A beta [1-40], and A beta [1-42]). A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was also utilized to compare levels of total soluble and insoluble A beta (1-40) and A beta (1-42) obtained from temporal cortex of MPS patients. Although no neurofibrillary tangles, senile plaques, or tau-positive lesions were detected in any of the MPS brains studied here, antibodies directed against A beta (1-40) intensely and diffusely stained the cytoplasm of cells throughout the brains of the MPS patients and the caprine MPS model. The ELISA assay also demonstrated a significant 3-fold increase in the level of soluble A beta (1-40) in the MPS brains compared with normal control brains. Thus, at least some of the metabolic defects that lead to accumulations of glycosaminoglycans in MPS also are associated with an increase in immunoreactive A beta (1-40) within the cytoplasmic compartment where they could contribute to the dysfunction and death of affected cells in these disorders, but not induce the formation of plaques and tangles. Models of MPS may enable mechanistic studies of the role A beta and glycosaminoglycans play in the amyloidosis that is a neuropathological feature of AD
— id: 24715, year: 1999, vol: 58, page: 815, stat: Journal Article,

RNA sequestration to pathological lesions of neurodegenerative diseases
Ginsberg, S D; Galvin, J E; Chiu, T S; Lee, V M; Masliah, E; Trojanowski, J Q
1998 Nov;96(5):487-494, Acta neuropathologica
Cytoplasmic RNA species have been identified recently within neurofibrillary tangles and senile plaques of Alzheimer's disease brain. To determine whether RNA sequestration is a common feature of other lesions found in progressive neurodegenerative disorders, acridine orange histofluorescence was employed, alone or in combination with immunohistochemistry and thioflavine-S staining to identify RNA species in paraffin-embedded brain tissue sections. Postmortem samples came from 39 subjects with the following diagnoses: Alzheimer's disease, amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam, corticobasal degeneration, diffuse Lewy body disease, normal controls, multiple system atrophy, Parkinson's disease, Pick's disease, progressive supranuclear palsy, and Shy-Drager syndrome. RNAs were detected in neurofibrillary tangles and neuritic senile plaques as well as in Pick bodies. However, Lewy bodies, Hirano bodies, and cytoplasmic glial inclusions did not contain abundant cytoplasmic RNA species. These observations demonstrate the selective localization of RNA species to distinct pathological lesions of neurodegenerative disease brains
— id: 110054, year: 1998, vol: 96, page: 487, stat: Journal Article,