Faculty Bibliography

Little is known about signaling mechanisms underlying temporal associative learning. Here, we show that mice with a targeted point mutation that prevents autophosphorylation of alphaCaMKII (alphaCaMKII(T286A)) learn trace eyeblink conditioning normally. This forms a sharp contrast to the severely impaired spatial learning in the water maze and contextual fear conditioning observed in alphaCaMKII(T286A) mutants. Importantly, hippocampal lesions impaired trace eyeblink conditioning in alphaCaMKII(T286A) mice, suggesting a potential role of hippocampal alphaCaMKII-independent mechanisms. These results indicate that hippocampal signaling mechanisms that underlie temporal associative learning as assessed by trace eyeblink conditioning may differ from those of spatial and contextual learning

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.

Increased numbers of autophagosomes/autophagic vacuoles are seen in a variety of physiological and pathological states in the nervous system. In many cases, it is unclear if this phenomenon is the result of increased autophagic activity or decreased autophagosome-lysosome fusion. The functional significance of autophagy and its relationship to cell death in the nervous system is also poorly understood. In this review, we have considered these issues in the context of acute neuronal injury and a range of chronic neurodegenerative conditions, including the Lurcher mouse, Alzheimer's, Parkinson's, Huntington's and prion diseases. While many issues remain unresolved, these conditions raise the possibility that autophagy can have either deleterious or protective effects depending on the specific situation and stage in the pathological process.

There is evidence that BDNF influences the birth of granule cells in the dentate gyrus, which is one of the few areas of the brain that demonstrates neurogenesis throughout life. However, studies to date have not examined this issue directly. To do so, we compared the effects of BDNF, phosphate-buffered saline (PBS), or bovine serum albumin (BSA) on neurogenesis after infusion into the hippocampus of the normal adult rat, using osmotic pumps that were implanted unilaterally in the dorsal hilus. BDNF, PBS, and BSA were infused for 2 weeks. The mitotic marker bromodeoxyuridine (BrdU) was administered twice daily during the 2-week infusion period. At least 1 month after infusion ended, brains were processed immunocytochemically using antibodies to BrdU, a neuronal nuclear protein (NeuN), or calbindin D28K (CaBP), which labels mature granule cells. Stereology was used to quantify BrdU-labeled cells in the dorsal hippocampus that were double-labeled with NeuN or CaBP. There was a statistically significant increase in BrdU(+)/NeuN(+) double-labeled cells in the granule cell layer after BDNF infusion relative to controls. The values for BrdU(+)/NeuN(+) cells were similar to BrdU(+)/CaBP(+) cells, indicating that most new neurons were likely to be granule cells. In addition, BrdU(+)/NeuN(+)-labeled cells developed in the hilar region after BDNF infusion, which have previously only been identified after severe continuous seizures (status epilepticus) and associated pathological changes. Remarkably, neurogenesis was also increased contralaterally, but BDNF did not appear to spread to the opposite hemisphere. Thus, infusion of BDNF to a local area can have widespread effects on hippocampal neurogenesis. The results demonstrate that BDNF administration to the dentate gyrus leads to increased neurogenesis of granule cells. They also show that ectopic granule cells develop after BDNF infusion, which suggests that ectopic migration is not necessarily confined to pathological conditions. These results are discussed in light of the evidence that BDNF increases neuronal activity in hippocampus. Thus, the mechanisms underlying neurogenesis following BDNF infusion could be due to altered activity as well as direct effects of BDNF itself, and this is relevant to studies of other growth factors because many of them have effects on neuronal excitability that are often not considered

Endocytosis is universally important in cell function. In the brain, the roles of endosomes are relatively more complex due to the unique polar morphology of neurons and specialized needs for inter-cellular communication. New evidence shows that endosome function is altered in a surprising range of neurodegenerative disorders, including in several inherited neurologic disorders where the causative mutations occur in genes that regulate endosome function. In Alzheimer's disease (AD), endosome abnormalities are among the earliest neuropathologic features to develop and have now been closely linked to genetic risk factors for AD, including APP triplication in Trisomy 21 (Down syndrome, DS) and ApoE4 genotype in sporadic AD. Recent findings on endosome regulation and developmental and late-onset neurodegenerative disease disorders are beginning to reveal how endocytic pathway impairment may lead to neuronal dysfunction and cell death in these disorders and may also promote amyloidogenesis in AD

The accumulation of lysosomes and their hydrolases within neurons is a well-established neuropathologic feature of Alzheimer disease (AD). Here we show that lysosomal pathology in AD brain involves extensive alterations of macroautophagy, an inducible pathway for the turnover of intracellular constituents, including organelles. Using immunogold labeling with compartmental markers and electron microscopy on neocortical biopsies from AD brain, we unequivocally identified autophagosomes and other prelysosomal autophagic vacuoles (AVs), which were morphologically and biochemically similar to AVs highly purified from mouse liver. AVs were uncommon in brains devoid of AD pathology but were abundant in AD brains particularly, within neuritic processes, including synaptic terminals. In dystrophic neurites, autophagosomes, multivesicular bodies, multilamellar bodies, and cathepsin-containing autophagolysosomes were the predominant organelles and accumulated in large numbers. These compartments were distinguishable from lysosomes and lysosomal dense bodies, previously shown also to be abundant in dystrophic neurites. Autophagy was evident in the perikarya of affected neurons, particularly in those with neurofibrillary pathology where it was associated with a relative depletion of mitochondria and other organelles. These observations provide the first evidence that macroautophagy is extensively involved in the neurodegenerative/regenerative process in AD. The striking accumulations of immature AV forms in dystrophic neurites suggest that the transport of AVs and their maturation to lysosomes may be impaired, thereby impeding the suspected neuroprotective functions of autophagy

This study confirms the presence of iron, co-localized with Abeta plaques, in PS/APP mouse brain, using Perls' stain for Fe3+ supplemented by 3,3'-diaminobenzidine (DAB) and Abeta immunohistochemistry in histological brains sections fixed with formalin or methacarn. In this study, the fixation process and the slice thickness did not interfere with the Perls' technique. The presence of iron in beta-amyloid plaques in PS/APP transgenic mice, a model of Alzheimer's disease (AD) pathology, may explain previous reports of reductions of transverse relaxation time (T2) in MRI studies and represent the source of the intrinsic Abeta plaque MR contrast in this model

The principal ovarian estrogen, estradiol, and brain-derived neurotrophic factor (BDNF) have widespread effects on the CNS that have usually been studied independently. This article examines the similarities in the effects of estradiol and BDNF in the hippocampus, in light of the evidence that estradiol can induce BDNF expression, and recent data suggesting that structural and electrophysiological effects of estradiol in the hippocampus might be mediated by BDNF. The possible role of BDNF as a signaling molecule downstream of estrogen in the hippocampus has implications for our understanding of several cellular and behavioral hippocampal functions, including dendritic and synaptic plasticity, learning and cognitive behavior. Furthermore, disruption of the relationship between estrogen and BDNF could contribute to neurological and psychiatric disorders that have been associated with the hippocampus, such as Alzheimer's disease, depression and epilepsy

Amyloid-containing tissue, whether from human patients or an animal model of a disease, is typically characterized by various biochemical and immunohistochemical techniques, many of which are described in detail in this volume. In this chapter, we describe a straightforward technique for the homogenization of tissue prior to these analyses. The technique is particularly well-suited for performing a large number of different biochemical analyses on a single mouse brain hemisphere. Starting with this homogenate, multiple characterizations can be done, including Western blot analysis and isolation of membrane-associated proteins, both of which are described here. Additional analyses can readily be performed on the tissue homogenate, including the ELISA quantitation of Abeta in the brain of a transgenic mouse model of beta-amyloid deposition. The ELISA technique is described in detail in the following chapter