Warren G. Young

Distribution of histone deacetylases 1-11 in the rat brain

Although protein phosphorylation has been characterized more extensively, modulation of the acetylation state of signaling molecules is now being recognized as a key means of signal transduction. The enzymes responsible for mediating these changes include histone acetyl transferases and histone deacetylases (HDACs). Members of the HDAC family of enzymes have been identified as potential therapeutic targets for diseases ranging from cancer to ischemia and neurode generation. We initiated a project to conduct comprehensive gene expression mapping of the 11 HDAC isoforms (HDAC1-11) (classes I, II, and IV) throughout the rat brain using high-resolution in situ hybridization (ISH) and imaging technology. Internal and external data bases were employed to identify the appropriate rat sequence information for probe selection. In addition, immunohistochemistry was performed on these samples to separately examine HDAC expression in neurons, astrocytes, oligodendrocytes, and endothelial cells in the CNS. This double-labeling approach enabled the identification of specific cell types in which the individual HDACs were expressed. The signals obtained by ISH were compared to radiolabeled standards and thereby enabled semiquantitative analysis of individual HDAC isoforms and defined relative levels of gene expression in >50 brain regions. This project produced an extensive atlas of 11 HDAC isoforms throughout the rat brain, including cell type localization, providing a valuable resource for examining the roles of specific HDACs in the brain and the development of future modulators of HDAC activity.

Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: A magnetic resonance microscopy and stereologic analysis

High-resolution magnetic resonance microscopy (MRM) was used to determine regional brain volumetric changes in a mouse model of Alzheimers disease. These transgenic (Tg) mice overexpress human mutant amyloid precursor protein (APP) V717F under control of platelet-derived growth factor promoter (PDAPP mice), and cortical and hippocampal β-amyloid (Aβ) deposits accumulate in heterozygotes after 8–10 mos. We used MRM to obtain 3D volumetric data on mouse brains imaged in their skulls to define genotype- and age-related changes. Hippocampal, cerebellar, and brain volumes and corpus callosum length were quantified in 40-, 100-, 365-, and 630-day-old mice. Measurements taken at age 100 days, before Aβ deposition, revealed a 12.3% reduction of hippocampus volume in Tg mice compared with WT controls. This reduction persisted without progression to age 21 mos. A significant 18% increase in hippocampal volume occurred between 40 and 630 days in WT mice, and no corresponding significant increase occurred in Tg mice. Cavalieri volume estimates of hippocampal subfields from 100-day-old Tg mice further localized a 28% volume deficit in the dentate gyrus. In addition, corpus callosum length was reduced by ≈25% in Tg mice at all ages analyzed. In summary, reduced hippocampal volume and corpus callosum length can be detected by MRM before Aβ deposition. We conclude that overexpression of APP and amyloid may initiate pathologic changes before the appearance of plaques, suggesting novel targets for the treatment of Alzheimers disease and further reinforcing the need for early diagnosis and treatment.

Neurochemical, morphologic, and laminar characterization of cortical projection neurons in the cingulate motor areas of the macaque monkey

The primate cingulate gyrus contains multiple cortical areas that can be distinguished by several neurochemical features, including the distribution of neurofilament protein‐enriched pyramidal neurons. In addition, connectivity and functional properties indicate that there are multiple motor areas in the cortex lining the cingulate sulcus. These motor areas were targeted for analysis of potential interactions among regional specialization, connectivity, and cellular characteristics such as neurochemical profile and morphology. Specifically, intracortical injections of retrogradely transported dyes and intracellular injection were combined with immunocytochemistry to investigate neurons projecting from the cingulate motor areas to the putative forelimb region of the primary motor cortex, area M1. Two separate groups of neurons projecting to area M1 emanated from the cingulate sulcus, one anterior and one posterior, both of which furnished commissural and ipsilateral connections with area M1. The primary difference between the two populations was laminar origin, with the anterior projection originating largely in deep layers, and the posterior projection taking origin equally in superficial and deep layers. With regard to cellular morphology, the anterior projection exhibited more morphologic diversity than the posterior projection. Commissural projections from both anterior and posterior fields originated largely in layer VI. Neurofilament protein distribution was a reliable tool for localizing the two projections and for discriminating between them. Comparable proportions of the two sets of projection neurons contained neurofilament protein, although the density and distribution of the total population of neurofilament protein‐enriched neurons was very different in the two subareas of origin. Within a projection, the participating neurons exhibited a high degree of morphologic heterogeneity, and no correlation was observed between somatodendritic morphology and neurofilament protein content. Thus, although the neurons that provide the anterior and posterior cingulate motor projections to area M1 differ morphologically and in laminar origin, their neurochemical profiles are similar with respect to neurofilament protein. This suggests that neurochemical phenotype may be a more important unifying feature for corticocortical projections than morphology.

Amyloid deposition in the hippocampus and entorhinal cortex: Quantitative analysis of a transgenic mouse model

Various transgenic mouse models of Alzheimers disease (AD) have been developed that overexpress mutant forms of amyloid precursor protein in an effort to elucidate more fully the potential role of β-amyloid (Aβ) in the etiopathogenesis of the disease. The present study represents the first complete 3D reconstruction of Aβ in the hippocampus and entorhinal cortex of PDAPP transgenic mice. Aβ deposits were detected by immunostaining and thioflavin fluorescence, and quantified by using high-throughput digital image acquisition and analysis. Quantitative analysis of amyloid load in hippocampal subfields showed a dramatic increase between 12 and 15 months of age, with little or no earlier detectable deposition. Three-dimensional reconstruction in the oldest brains visualized previously unrecognized sheets of Aβ coursing through the hippocampus and cerebral cortex. In contrast with previous hypotheses, compact plaques form before significant deposition of diffuse Aβ, suggesting that different mechanisms are involved in the deposition of diffuse amyloid and the aggregation into plaques. The dentate gyrus was the hippocampal subfield with the greatest amyloid burden. Sublaminar distribution of Aβ in the dentate gyrus correlated most closely with the termination of afferent projections from the lateral entorhinal cortex, mirroring the selective vulnerability of this circuit in human AD. This detailed temporal and spatial analysis of Aβ and compact amyloid deposition suggests that specific corticocortical circuits express selective, but late, vulnerability to the pathognomonic markers of amyloid deposition, and can provide a basis for detecting prior vulnerability factors.

Differential vulnerability of oculomotor, facial, and hypoglossal nuclei in G86R superoxide dismutase transgenic mice

In recent years, several mouse models of amyotrophic lateral sclerosis (ALS) have been developed. One, caused by a G86R mutation in the superoxide dismutase‐1 (SOD‐1) gene associated with familial ALS, has been subjected to extensive quantitative analyses in the spinal cord. However, the human form of ALS includes pathology elsewhere in the nervous system. In the present study, analyses were extended to three motor nuclei in the brainstem. Mutant mice and control littermates were evaluated daily, and mutants, along with their littermate controls, were killed when they were severely affected. Brains were removed after perfusion and processed for Nissl staining, the samples were randomized, and the investigators were blinded to their genetic status. Stereologic methods were used to estimate the number of neurons, mean neuronal volumes, and nuclear volume in three brainstem motor nuclei known to be differetially involved in the human form of the disease, the oculomotor, facial, and hypoglossal nuclei. In the facial nucleus, neuron number consistently declined (48%), an effect that was correlated with disease severity. The nuclear volume of the facial nucleus was smaller in the SOD‐1 mutant mice (45.7% difference from control mice) and correlated significantly with neuron number. The oculomotor and hypoglossal nuclei showed less extreme involvement (<10% neuronal loss overall), with a trend toward fewer neurons in the hypoglossal nucleus of animals with severe facial nucleus involvement. In the oculomotor nucleus, neuronal loss was seen only once in five mice, associated with very severe disease. There was no significant change in the volume of individual neurons in any of these three nuclei in any transgenic mouse. These results suggest that different brainstem motor nuclei are differentially affected in this SOD‐1 mutant model of ALS. The relatively moderate and late involvement of the hypoglossal nucleus indicates that, although the general patterns of neuronal pathology match closely those seen in ALS patients, some differences exist in this transgenic model compared with the progression of the disease in humans. However, these patterns of cellular vulnerability may provide clues for understanding the differential susceptibility of neural structures in ALS and other neurodegenerative diseases. J. Comp. Neurol. 416:112–125, 2000.

Numbers of Meynert and layer IVB cells in area V1: A stereologic analysis in young and aged macaque monkeys

Visual impairments that are not related to optical changes are not uncommon during aging, and a number of psychophysical investigations have documented deficits in motion detection as well as in spatiotemporal contrast sensitivity in elderly people. However, little is known about the extent and nature of age‐related changes in neural structure and how they may affect visual function in aging. To address this question, the authors analyzed the effect of aging on two well‐characterized neuronal populations in the primary visual cortex (area V1) of macaque monkeys. Four young adult (ages, 7–11 years) and four aged (ages, 26–32 years) rhesus monkeys were analyzed. The animals were perfused, and their brains were prepared for immunohistochemistry with an antibody to neurofilament protein. Unbiased stereologic estimates of the total numbers of neurofilament protein‐containing layer IVB cells and Meynert cells were obtained by using the optical fractionator method for the calcarine cortex and the opercular cortex separately. Stereologic estimates of the volume of these parts of area V1 also were calculated by using the Cavalieri principle. A considerable degree of interindividual variability in neuron numbers and cortical volume was observed among animals of both groups. However, there were no differences in either Meynert cell numbers or layer IVB cell numbers between the aged group and the young group. It is noteworthy that the oldest animal in the sample had the lowest numbers of Meynert cells, indicating that, despite the small size of the available sample, it is possible that some animals have a certain degree of neuronal loss in area V1 during aging. No change in the volume of area V1 was observed as a function of aging. These data suggest that the deficits that occur during aging in the visual system are not due to the loss of highly specific neocortical neuronal populations, such as those analyzed in this study. Rather, it is possible that more subtle alterations in the neurochemical characteristics or synaptic organization of the functional pathways subserving the different visual modalities are responsible for these deficits. J. Comp. Neurol. 420:113–126, 2000.

Effects of social deprivation in prepubescent rhesus monkeys: immunohistochemical analysis of the neurofilament protein triplet in the hippocampal formation

Social deprivation during early postnatal life has profound and long-lasting effects on the behavior of primates, including prolonged and exaggerated responses to stress as well as impaired performance on a variety of learning tasks. Although the cellular changes that underlie such alterations in behavior are unknown, environmentally induced psychopathology may involve morphologic or biochemical changes in select neuronal populations. The hippocampal formation of both socially deprived and socially reared prepubescent rhesus monkeys was selected for immunocytochemical investigation because of its association with the behavioral stress response and learning. Immunocytochemical analysis using antibodies specific for the neurofilament protein triplet was performed since these proteins are modified within degenerating neurons in a variety of neurodegenerative disorders. Results from optical density measurements indicate an increase in the intensity of non-phosphorylated neurofilament protein immunoreactivity in the dentate gyrus granule cell layer of socially deprived monkeys in comparison with that of socially reared animals, suggesting that early social deprivation may result in an increase in the amount of non-phosphorylated neurofilament protein in these cells. This phenotypic difference in dentate granule cells between differentially reared monkeys supports the notion that specific subpopulations of neurons in brain regions that subserve complex behaviors may undergo long-term modifications induced by environmental conditions. Furthermore, the data suggest that constitutive chemical components related to structural integrity may be as susceptible to early environmental manipulations as the more traditionally viewed measures of cellular perturbations, such as neurotransmitter dynamics, cell density and the establishment of connectivity. The observed modifications may serve as an anatomical substrate for behavioral abnormalities that persist in later life.

Cortical neuronal cytoskeletal changes associated with FIV infection

HIV-1 infection is often complicated by central nervous system (CNS) dysfunction. Degenerative neuronal changes as well as neuronal loss have been documented in individuals with AIDS. Feline immunodeficiency virus (FIV) infection of cats provides a model for both the immune and the central nervous system manifestations of HIV infection of humans. In this study we have examined neurons in the frontal cortex of feline immunodeficiency virus-infected cats and controls for immunoreactivity with SMI 32, an antibody recognizing a non-phosphorylated epitope on neurofilaments. We noted a significant increase in the number of immunoreactive pyramidal cells in infected animals compared to controls. The changes seen in the neuronal cytoskeleton as a consequence of the inoculation with FIV were similar to those seen in humans undergoing the normal aging process as well as those suffering from neurological diseases, including Alzheimers and dementia pugilistica. The changes we noted in the feline brain were also similar to that reported in animals with traumatic injuries or with spontaneously occurring or induced motor neuron diseases, suggesting that the increase in reactivity represents a deleterious effect of FIV on the central nervous system.

Standardized quantitative in situ hybridization using radioactive oligonucleotide probes for detecting relative levels of mRNA transcripts verified by real-time PCR

In situ hybridization (ISH) is an essential technique for mapping gene expression in the brain. Although many ISH protocols provide for quantitative analysis of individual mRNAs in different brain regions or across experimental conditions, this technique has lacked the necessary standardization for quantitative comparisons between different mRNA transcripts. We have developed a standardized quantitative ISH (SQuISH) protocol that utilizes multiple radioactive oligonucleotide probes, providing for increased sensitivity, decreased background and accurate comparison of relative mRNA levels. We evaluated the SQuISH protocol against a riboprobe-based ISH procedure by comparing the mRNA expression levels in the brain for two transcripts, insulin receptor substrate p53 (IRSp53) and Calsenilin. The results of these two methods were then validated by real-time quantitative PCR. Both protocols exhibited identical mRNA expression patterns for IRSp53 and Calsenilin. In three brain regions analyzed, the levels of IRSp53 mRNA expression were approximately 1.5-fold higher with the riboprobe-based ISH than with the SQuISH procedure, although the relative abundance in regional expression levels was similar between the two methods. In contrast, the levels of Calsenilin mRNA expression were 10-17-fold higher with the riboprobe-based ISH than with the SQuISH procedure and the relative abundance in regional expression levels was different. When compared to the real-time PCR results, the SQuISH trade mark method showed almost identical relative levels of IRSp53 to Calsenilin mRNA in all three brain regions analyzed, while the riboprobe-based procedure showed a completely opposite trend. These results support the accuracy of the SQuISH protocol for determining relative mRNA levels in the brain.

New Prospects and Strategies for Drug Target Discovery in Neurodegenerative Disorders

SummaryThe future of neurodegenerative therapeutics development depends upon effective disease modification strategies centered on carefully investigated targets. Pharmaceutical research endeavors that probe for a much deeper understanding of disease pathogenesis, and explain how adaptive or compensatory mechanisms might be engaged to delay disease onset or progression, will produce the needed breakthroughs. Below, we discuss the prospects for new targets emerging out of the study of brain disease genes and their associated pathogenic pathways. We describe a general experimental paradigm that we are employing across several mouse models of neurodegenerative disease to elucidate molecular determinants of selective neuronal vulnerability. We outline key elements of our target discovery program and provide examples of how we integrate genomic technologies, neuroanatomical methods, and mouse genetics in the search for neurodegenerative disease targets.