Theresa Schuck

PP2A mRNA Expression Is Quantitatively Decreased in Alzheimer's Disease Hippocampus ☆

Since abnormal tau phosphorylation may play a role in neurofibrillary tangle (NFT) formation in aging and Alzheimers disease (AD), we probed the distribution and abundance of protein phosphatase 2A (PP2A) catalytic (Calpha) and regulatory (PR55alpha and gamma, PR61varepsilon and delta) subunit mRNA in control and AD hippocampus using in situ hybridization. Quantitation of grain density per neuron area of PP2A subunits and beta-actin was determined for the CA3 region of hippocampus and cerebellum, while a qualitative assessment was performed for CA1, CA4, and dentate gyrus. All subunits are expressed in neurons, while PR55gamma and PR55alpha mRNA are also evident in glia. The expression levels of Calpha, all PP2A regulatory subunits studied, and beta-actin were similar in control and AD cerebellum. beta-Actin mRNA was, however, reduced in AD hippocampus. In addition to the generalized reduction of mRNA, as indicated by decreased beta-actin signal, there was a significant loss of Calpha, PR55gamma, and PR61epsilon mRNA in the CA3 hippocampus of AD. This study delineates the distribution of critical PP2A mRNAs and reveals a neuron- and subunit-specific reduction in PP2A catalytic and regulatory mRNA in AD hippocampus. This could result in decreased protein expression and phosphatase activity, leading to the hyperphosphorylation of tau and the formation of NFTs, as well as neuron degeneration in AD.

Distribution of tau proteins in the normal human central and peripheral nervous system.

In human brain, antibodies to tau proteins primarily label abnormal rather than normal structures. This might reflect altered immunoreactivity owing to post-mortem proteolysis, disease, or species differences. We addressed this issue by comparing the distribution of tau in bovine and human post-mortem nervous system tissues and in human neural cell lines, using new monoclonal antibodies (MAb) specific for phosphate-independent epitopes in bovine and human tau. In neocortex, hippocampus, and cerebellum, immunoreactive tau was widely expressed but segregated into the axon-neuropil domain of neurons. In spinal cord and peripheral nervous system, tau immunoreactivity was similarly segregated but less abundant. No immunoreactive tau was detected with our MAb in glial cells or in human neural cell lines that express neurofilament or glial filament proteins. Post-mortem delays in tissue denaturation of less than 24 hr did not affect the distribution of tau, but the method used to denature tissues did, i.e., microwave treatment preserved tau immunoreactivity more effectively than chemical fixatives such as Bouins solution, and formalin-fixed tissue samples reacted poorly with our anti-tau MAb. We conclude that the distribution of tau proteins in human nervous system is similar to that described in perfusion-fixed experimental animals, and that visualization of normal immunoreactive tau in human tissues is critically dependent on the procedures used to denature post-mortem tissue samples. Furthermore, microenvironmental factors in different neuroanatomical sites may affect the regional expression of tau.

Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis

Abnormal TDP-43 aggregation is a prominent feature in the neuropathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Mutations in TARDBP, the gene encoding TDP-43, cause some cases of ALS. The normal function of TDP-43 remains incompletely understood. To better understand TDP-43 biology, we generated mutant mice carrying a genetrap disruption of Tardbp. Mice homozygous for loss of TDP-43 are not viable. TDP-43 deficient embryos die about day 7.5 of embryonic development thereby demonstrating that TDP-43 protein is essential for normal prenatal development and survival. However, heterozygous Tardbp mutant mice exhibit signs of motor disturbance and muscle weakness. Compared with wild type control littermates, Tardbp+/− animals have significantly decreased forelimb grip strength and display deficits in a standard inverted grid test despite no evidence of pathologic changes in motor neurons. Thus, TDP-43 is essential for viability, and mild reduction in TDP-43 function is sufficient to cause motor deficits without degeneration of motor neurons.

Differential Expression and Distribution of α-, β-, and γ-Synuclein in the Developing Human Substantia Nigra

Abstract Although the functions of α-, β-, and γ-synuclein (αS, βS, γS, respectively) are unknown, these synaptic proteins are implicated in the pathogenesis of Parkinsons disease (PD) and related disorders. For example, αS forms Lewy bodies (LBs) in substantia nigra (SN) neurons of PD. However, since it is not known how these hallmark PD lesions contribute to the degeneration of SN neurons or what the normal function of αS is in SN neurons, we studied the developing human SN from 11 weeks gestational age (GA) to 16 years of age using immunohistochemistry and antibodies to αS, βS, γS, other synaptic proteins, and tyrosine hydoxylase (TH). SN neurons expressed TH at 11 weeks GA and αS, βS, and γS appeared initially at 15, 17, and 18 weeks GA, respectively. These synucleins first appeared in perikarya of SN neurons after synaptophysin, but about the same time as synaptotagmin and synaptobrevin. Redistribution of αS from perikarya to processes of SN neurons occurred by 18 weeks GA in parallel with synaptophysin, while βS and synaptotagmin were redistributed similarly between 20 and 28 weeks GA and this also occurred with γS and synaptobrevin between 33 weeks GA and 9 months postnatal. These data suggest that αS, βS, and γS may play a functional role in the development and maturation of SN neurons, but it remains to be determined how sequestration of αS as LBs in PD contributes to the degeneration of SN neurons.

The Fluorescent Congo Red Derivative, (Trans, Trans)−1-Bromo-2,5-Bis-(3-Hydroxycarbonyl-4-Hydroxy)Styrylbenzene (BSB), Labels Diverse β-Pleated Sheet Structures in Postmortem Human Neurodegenerative Disease Brains

A novel Congo red-derived fluorescent probe (trans, trans),-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) that binds to amyloid plaques of postmortem Alzheimers disease brains and in transgenic mouse brains in vivo was designed as a prototype imaging agent for Alzheimers disease. In the current study, we used BSB to probe postmortem tissues from patients with various neurodegenerative diseases with diagnostic lesions characterized by fibrillar intra- or extracellular lesions and compared these results with standard histochemical dyes such as thioflavin S and immunohistochemical stains specific for the same lesions. These data show that BSB binds not only to extracellular amyloid beta protein, but also many intracellular lesions composed of abnormal tau and synuclein proteins and suggests that radioiodinated BSB derivatives or related ligands may be useful imaging agents to monitor diverse amyloids in vivo.

A platform for discovery: The University of Pennsylvania Integrated Neurodegenerative Disease Biobank

Neurodegenerative diseases (NDs) are defined by the accumulation of abnormal protein deposits in the central nervous system (CNS), and only neuropathological examination enables a definitive diagnosis. Brain banks and their associated scientific programs have shaped the actual knowledge of NDs, identifying and characterizing the CNS deposits that define new diseases, formulating staging schemes, and establishing correlations between neuropathological changes and clinical features. However, brain banks have evolved to accommodate the banking of biofluids as well as DNA and RNA samples. Moreover, the value of biobanks is greatly enhanced if they link all the multidimensional clinical and laboratory information of each case, which is accomplished, optimally, using systematic and standardized operating procedures, and in the framework of multidisciplinary teams with the support of a flexible and user‐friendly database system that facilitates the sharing of information of all the teams in the network. We describe a biobanking system that is a platform for discovery research at the Center for Neurodegenerative Disease Research at the University of Pennsylvania.

Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues

In contrast with results obtained in experimental animals, antibodies to microtubule associated protein-2 (MAP2) preferentially label abnormal structures in human nervous system tissue samples, but the normal sites at which MAP2 is expressed are not well-defined. To determine the distribution of MAP2 in the human central (CNS) and peripheral (PNS) nervous systems, we prepared monoclonal antibodies (MAbs) specific to MAP2, and compared the localization of this MAP in postmortem bovine and human tissues as well as in several human neural cell lines that express either neurofilament (NF) or glial filament (GF) proteins. Eight MAbs specific for phosphate-independent epitopes in bovine and human MAP2 were obtained, and those that performed well in tissues produced immunoreactivity confined to the somatodendritic domain of neurons in bovine and human CNS and PNS tissues. Other neural cells (e.g. astrocytes) did not express MAP2 immunoreactivity using these MAbs. Postmortem delays of less than 24 h prior to tissue denaturation did not affect the distribution of MAP2 immunoreactivity. However, microwave denaturation of these tissues preserved MAP2 immunoreactivity better than fixation with Bouins solution or formalin. Microwave treatment also improved the immunoreactivity of several MAbs for NF and GF proteins. Finally, MAP2 was not detected in human neural cell lines that express NF (2) or GF (1) proteins. We conclude that microwave denaturation provides an effective means to preserve the immunoreactivity of normal human neuronal cytoskeletal proteins, and that this method of tissue denaturation allows the normal distribution of MAP2 to be defined in postmortem samples of human CNS and PNS tissues.

Amyloid plaques in Guam amyotrophic lateral sclerosis/ parkinsonism-dementia complex contain species of Aβ similar to those found in the amyloid plaques of Alzheimer’s disease and pathological aging

Abstract The Guamanian amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is characterized by abundant neurofibrillary pathology and neuron loss. In contrast to Alzheimer’s disease (AD), where extensive neurofibrillary lesions always occur with deposits of Aβ in numerous amyloid plaques, Aβ-rich amyloid plaques are absent or rare in most ALS/PDC patients. To characterize the amyloid plaques in the latter patients, we probed plaque-rich sections of their brains by immunohistochemistry using well-characterized antibodies to specific epitopes in the N and C termini of Aβ as well as to defined epitopes in hyperphosphorylated tau (PHFtau). The results indicate that the species of Aβ in the amyloid plaques of ALS/PDC patients resemble those detected in the amyloid plaques of cognitively intact subjects with pathological aging as well as patients with AD. However, the paucity of PHFtau-positive neurites in the ALS/PDC plaques suggests that they reflect pathological aging rather than AD.

Acalculia in Autopsy-Proven Corticobasal Degeneration

Corticobasal degeneration (CBD) is a neurodegenerative condition presenting with an asymmetric extrapyramidal disorder, cortical sensory loss, and apraxia. While the original case descriptions mentioned acalculia,1 few studies have investigated this,2,3 and reports of acalculia in autopsy-proven CBD are very rare. We detail 2 autopsy-defined CBD cases with acalculia to emphasize that CBD compromises cognitive functioning due to disease that includes parietal cortex. ### Case 1. A 72-year-old right-handed woman with hypertension and hypothyroidism was evaluated for progressive cognitive and motor difficulties over 3 years. She first noted writing difficulty. Her right hand began performing involuntary, semi-purposeful movements. She required increasing assistance dressing and cutting food. She misjudged spatial relationships while driving and cooking. She had several falls. Examination revealed Mini-Mental State Examination (MMSE) score of 27. She had ideomotor apraxia, slowed writing, and difficulty copying geometric designs. Number knowledge was impaired, including miscounting “X” marks on a paper and erring during oral and written calculations (e.g., given “9 + 12,” she responded “20”). Memory, digit span, reading, comprehension, and speech were intact. She had axial rigidity and decreased right arm swing, but no other involuntary movements. Neuropsychological evaluation ( z scores relative to 25 demographically matched controls) revealed deficits on spatial tasks (e.g., geometric figure copy z = −7.74), mild executive dysfunction (e.g., animal category naming fluency z = −1.90), and preserved language (e.g., Boston Naming test z = −0.86) and memory (delayed recall of a 10-word list z = 0.02). MRI showed parietal atrophy. Over the next 18 months, ideomotor apraxia worsened, and she …

Characterizing the human hippocampus in aging and Alzheimer’s disease using a computational atlas derived from ex vivo MRI and histology

Significance There has been increasing interest in hippocampal subfield morphometry in aging and disease using in vivo MRI. However, research on in vivo morphometry is hampered by the lack of a definitive reference model describing regional effects of aging and disease pathology on the hippocampus. To address this limitation, we built a 3D probabilistic atlas of the hippocampus combining postmortem MRI with histology, allowing us to investigate Alzheimer’s disease (AD)-related effects on hippocampal subfield morphometry, derived from histology. Our results support the hypothesis of differential involvement of hippocampal subfields in AD, providing further impetus for more granular study of the hippocampus in aging and disease during life. Furthermore, this atlas provides an important anatomical reference for hippocampal subfield research. Although the hippocampus is one of the most studied structures in the human brain, limited quantitative data exist on its 3D organization, anatomical variability, and effects of disease on its subregions. Histological studies provide restricted reference information due to their 2D nature. In this paper, high-resolution (∼200 × 200 × 200 μm3) ex vivo MRI scans of 31 human hippocampal specimens are combined using a groupwise diffeomorphic registration approach into a 3D probabilistic atlas that captures average anatomy and anatomic variability of hippocampal subfields. Serial histological imaging in 9 of the 31 specimens was used to label hippocampal subfields in the atlas based on cytoarchitecture. Specimens were obtained from autopsies in patients with a clinical diagnosis of Alzheimers disease (AD; 9 subjects, 13 hemispheres), of other dementia (nine subjects, nine hemispheres), and in subjects without dementia (seven subjects, nine hemispheres), and morphometric analysis was performed in atlas space to measure effects of age and AD on hippocampal subfields. Disproportional involvement of the cornu ammonis (CA) 1 subfield and stratum radiatum lacunosum moleculare was found in AD, with lesser involvement of the dentate gyrus and CA2/3 subfields. An association with age was found for the dentate gyrus and, to a lesser extent, for CA1. Three-dimensional patterns of variability and disease and aging effects discovered via the ex vivo hippocampus atlas provide information highly relevant to the active field of in vivo hippocampal subfield imaging.