Faculty Bibliography

Alzheimer's disease (AD) and epilepsy are separated in the medical community, but seizures occur in some patients with AD, and AD is a risk factor for epilepsy. Furthermore, memory impairment is common in patients with epilepsy. The relationship between AD and epilepsy remains an important question because ideas for therapeutic approaches could be shared between AD and epilepsy research laboratories if AD and epilepsy were related. Here we focus on one of the many types of epilepsy, temporal lobe epilepsy (TLE), because patients with TLE often exhibit memory impairment, depression and other comorbidities that occur in AD. Moreover, the seizures that occur in patients with AD may be nonconvulsive, which occur in patients with TLE. Here we first compare neuropathology in TLE and AD with an emphasis on the hippocampus, which is central to both AD and TLE research. Then we compare animal models of AD pathology with animal models of TLE. Although many aspects of the comparisons are still controversial, there is one conclusion that we suggest is clear: some animal models of TLE could be used to help address questions in AD research, and some animal models of AD pathology are bona fide animal models of epilepsy.

Thyroid hormone is critical for central nervous system development. Fetal hypothyroidism leads to reduced cognitive performance in offspring as well as other effects on neural development in both humans and experimental animals. The nature of these impairments suggests that thyroid hormone may exert its effects via dysregulation of the neurotrophin brain-derived neurotrophic factor (BDNF), which is critical to normal development of the central nervous system and has been implicated in neurodevelopmental disorders. The only evidence of BDNF dysregulation in early development, however, comes from experimental models in which severe prenatal hypothyroidism occurred. By contrast, milder prenatal hypothyroidism has been shown to alter BDNF levels and BDNF-dependent functions only much later in life. We hypothesized that mild experimental prenatal hypothyroidism might lead to dysregulation of BDNF in the early postnatal period. BDNF levels were measured by ELISA at 3 or 7 d after birth in different regions of the brains of rats exposed to propylthiouracil (PTU) in the drinking water. The dose of PTU that was used induced mild maternal thyroid hormone insufficiency. Pups, but not the parents, exhibited alterations in tissue BDNF levels. Hippocampal BDNF levels were reduced at both d 3 and 7, but no significant reductions were observed in either the cerebellum or brain stem. Unexpectedly, more males than females were born to PTU-treated dams, suggesting an effect of PTU on sex determination. These results support the hypothesis that reduced hippocampal BDNF levels during early development may contribute to the adverse neurodevelopmental effects of mild thyroid hormone insufficiency during pregnancy.

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

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

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

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

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. Emerging evidence indicates that amyloid-beta (Abeta) peptides enter mitochondria and may thereby disrupt mitochondrial function in brains of AD patients and transgenic model mice. However, it remains to be determined whether the beta-cleaved C-terminal fragment (C99), another neurotoxic fragment of amyloid precursor protein (APP), may accumulate in mitochondria of neurons affected by AD. Using immunoblotting, digitonin fractionation and immunofluorescence labeling techniques, we found that C99 is targeted to mitochondria, in particular, to the mitoplast (i.e., inner membrane and matrix compartments) in brains of AD transgenic mice (5XFAD model). Furthermore, full-length APP (fl-APP) was also identified in mitochondrial fractions of 5XFAD mice. Remarkably, partial deletion of the beta-site APP-cleaving enzyme 1 (BACE1(+/-)) almost completely abolished mitochondrial targeting of C99 and fl-APP in 5XFAD mice at 6 months of age. However, substantial amounts of C99 and fl-APP accumulation remained in mitochondria of 12-month-old BACE1(+/-).5XFAD mouse brains. Consistent with these changes in mitochondrial C99/fl-APP levels, BACE1(+/-) deletion age-dependently rescued mitochondrial dysfunction in 5XFAD mice, as assessed by cytochrome c release from mitochondria, reduced redox or complex activities and oxidative DNA damage. Moreover, BACE1(+/-) deletion also improved memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at 6 months but not at 12 months of age. Taken together, our findings suggest that mitochondrial accumulation of C99 and fl-APP may occur through BACE1-dependent mechanisms and contribute to inducing mitochondrial dysfunction and cognitive impairments associated with AD.

Increasing evidence suggests that reductions in brain-derived neurotrophic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB) may have a role in the pathogenesis of Alzheimer's disease (AD). However, the efficacy and safety profile of BDNF therapy (eg, gene delivery) remains to be established toward clinical trials. Here, we evaluated the effects of 7,8-dihydroxyflavone (7,8-DHF), a recently identified small-molecule TrkB agonist that can pass the blood-brain barrier, in the 5XFAD transgenic mouse model of AD. 5XFAD mice at 12-15 months of age and non-transgenic littermate controls received systemic administration of 7,8-DHF (5 mg/kg, i.p.) once daily for 10 consecutive days. We found that 7,8-DHF rescued memory deficits of 5XFAD mice in the spontaneous alternation Y-maze task. 5XFAD mice showed impairments in the hippocampal BDNF-TrkB pathway, as evidenced by significant reductions in BDNF, TrkB receptors, and phosphorylated TrkB. 7,8-DHF restored deficient TrkB signaling in 5XFAD mice without affecting endogenous BDNF levels. Meanwhile, 5XFAD mice exhibited elevations in the beta-secretase enzyme (BACE1) that initiates amyloid-beta (Abeta) generation, as observed in sporadic AD. Interestingly, 7,8-DHF blocked BACE1 elevations and lowered levels of the beta-secretase-cleaved C-terminal fragment of amyloid precursor protein (C99), Abeta40, and Abeta42 in 5XFAD mouse brains. Furthermore, BACE1 expression was decreased by 7,8-DHF in wild-type mice, suggesting that BDNF-TrkB signaling is also important for downregulating baseline levels of BACE1. Together, our findings indicate that TrkB activation with systemic 7,8-DHF can ameliorate AD-associated memory deficits, which may be, at least in part, attributable to reductions in BACE1 expression and beta-amyloidogenesis.

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.

Neuronal cell culture models have been used to demonstrate the protective effects of cystatin C against a variety of insults, including the toxicity induced by oligomeric and fibrillar amyloid beta (Abeta). Here, we describe assays quantifying cystatin C protective effects against cytotoxicity induced by nutrient deprivation, oxidative stress, or cytotoxic forms of Abeta. Three methods for the evaluation of either cell death or cell survival are described: measurement of metabolic activity, cell death, and cell division. The cell culture models used are murine primary cortical neurons and murine primary cerebral smooth muscle cells. The effects of exogenously applied cystatin C are studied by comparing the viability of nonstressed control, stressed control, and cystatin C-treated stressed cells. The effect of endogenous level of cystatin C expression is studied by comparing stressed primary cells isolated from brains of cystatin C transgenic, cystatin C knockout, and wild-type mice.