Faculty Bibliography

This review describes developments in epilepsy research during the last 3 to 4 decades that focused on the dentate gyrus (DG) and its role in temporal lobe epilepsy (TLE). The emphasis is on basic research in laboratory animals and is chronological, starting with hypotheses that attracted a lot of attention in the 1980s. Then experiments are described that addressed the questions, as well as new methods that often made the experiments possible. In addition, where new questions arose and the implications for clinical epilepsy are discussed.

Choline is critical for normative function of 3 major pathways in the brain, including acetylcholine biosynthesis, being a key mediator of epigenetic regulation, and serving as the primary substrate for the phosphatidylethanolamine N-methyltransferase pathway. Sufficient intake of dietary choline is critical for proper brain function and neurodevelopment. This is especially important for brain development during the perinatal period. Current dietary recommendations for choline intake were undertaken without critical evaluation of maternal choline levels. As such, recommended levels may be insufficient for both mother and fetus. Herein, we examined the impact of perinatal maternal choline supplementation (MCS) in a mouse model of Down syndrome and Alzheimer's disease, the Ts65Dn mouse relative to normal disomic littermates, to examine the effects on gene expression within adult offspring at ∼6 and 11 mo of age. We found MCS produces significant changes in offspring gene expression levels that supersede age-related and genotypic gene expression changes. Alterations due to MCS impact every gene ontology category queried, including GABAergic neurotransmission, the endosomal-lysosomal pathway and autophagy, and neurotrophins, highlighting the importance of proper choline intake during the perinatal period, especially when the fetus is known to have a neurodevelopmental disorder such as trisomy.-Alldred, M. J., Chao, H. M., Lee, S. H., Beilin, J., Powers, B. E., Petkova, E., Strupp, B. J., Ginsberg, S. D. Long-term effects of maternal choline supplementation on CA1 pyramidal neuron gene expression in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Young adult-born granule cells (abGCs) in the dentate gyrus (DG) have a profound impact on cognition and mood. However, it remains unclear how abGCs distinctively contribute to local DG information processing. We found that the actions of abGCs in the DG depend on the origin of incoming afferents. In response to lateral entorhinal cortex (LEC) inputs, abGCs exert monosynaptic inhibition of mature granule cells (mGCs) through group II metabotropic glutamate receptors. By contrast, in response to medial entorhinal cortex (MEC) inputs, abGCs directly excite mGCs through N-methyl-d-aspartate receptors. Thus, a critical function of abGCs may be to regulate the relative synaptic strengths of LEC-driven contextual information versus MEC-driven spatial information to shape distinct neural representations in the DG.

BACKGROUND:.
 CONCLUSIONS: Using novel and highly specific antibodies in combination with electron microscopy may reveal important information about the timing of aberrant protein accumulation, as well as the progression of abnormalities in the endolysosomal systems that sort and clear these peptides.

Hippocampal CA1 pyramidal neurons, a major component of the medial temporal lobe memory circuit, are selectively vulnerable during the progression of Alzheimer's disease (AD). The cellular mechanism(s) underlying degeneration of these neurons and the relationship to cognitive performance remains largely undefined. Here, we profiled neurotrophin and neurotrophin receptor gene expression within microdissected CA1 neurons along with regional hippocampal dissections from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD using laser capture microdissection (LCM), custom-designed microarray analysis, and qPCR of CA1 subregional dissections. Gene expression levels were correlated with cognitive test scores and AD neuropathology criteria. We found a significant downregulation of several neurotrophin genes (e.g., Gdnf, Ngfb, and Ntf4) in CA1 pyramidal neurons in MCI compared to NCI and AD subjects. In addition, the neurotrophin receptor transcripts TrkB and TrkC were decreased in MCI and AD compared to NCI. Regional hippocampal dissections also revealed select neurotrophic gene dysfunction providing evidence for vulnerability within the hippocampal proper during the progression of dementia. Downregulation of several neurotrophins of the NGF family and cognate neurotrophin receptor (TrkA, TrkB, and TrkC) genes correlated with antemortem cognitive measures including the Mini-Mental State Exam (MMSE), a composite global cognitive score (GCS), and Episodic, Semantic, and Working Memory, Perceptual Speed, and Visuospatial domains. Significant correlations were found between select neurotrophic expression downregulation and neuritic plaques (NPs) and neurofibrillary tangles (NFTs), but not diffuse plaques (DPs). The data suggest that dysfunction of neurotrophin signaling complexes have profound negative sequelae within vulnerable hippocampal cell types, which play a role in mnemonic and executive dysfunction during the progression of AD.

Autophagy-lysosome pathway (ALP) disruption is considered pathogenic in multiple neurodegenerative diseases; however, current methods are inadequate to investigate macroautophagy/autophagy flux in brain in vivo and its therapeutic modulation. Here, we describe a novel autophagy reporter mouse (TRGL6) stably expressing a dual-fluorescence-tagged LC3 (tfLC3, mRFP-eGFP-LC3) by transgenesis selectively in neurons. The tfLC3 probe distributes widely in the central nervous system, including spinal cord. Expression levels were similar to endogenous LC3 and induced no detectable ALP changes. This ratiometric reporter registers differential pH-dependent changes in color as autophagosomes form, fuse with lysosomes, acidify, and degrade substrates within autolysosomes. We confirmed predicted changes in neuronal autophagy flux following specific experimental ALP perturbations. Furthermore, using a third fluorescence label in TRGL6 brains to identify lysosomes by immunocytochemistry, we validated a novel procedure to detect defective autolysosomal acidification in vivo. Thus, TRGL6 mice represent a unique tool to investigate in vivo ALP dynamics in specific neuron populations in relation to neurological diseases, aging, and disease modifying agents. Abbreviations: ACTB: actin, beta; AD: Alzheimer disease; AL: autolysosomes; ALP: autophagy-lysosome pathway; AP: autophagosome; APP: amyloid beta (Abeta) precursor protein; ATG5: autophagy related 5; ATG7: autophagy related 7; AV: autophagic vacuoles; CNS: central nervous system; CTSD: cathepsin D; CQ: chloroquine; DMEM: Dulbecco's modified Eagle's medium; GFP: green fluorescent protein; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2/GATE16: gamma-aminobutyric acid (GABA) receptor-associated protein-like 2; ICC: immunocytochemistry; ICV: intra-cerebroventricular; LAMP2: lysosomal-associated membrane protein 2; Leup: leupeptin; LY: lysosomes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RFP: red fluorescent protein; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; tfLC3: mRFP-eGFP-LC3; TRGL6: Thy1 mRFP eGFP LC3-line 6; PCR: polymerase chain reaction; PD: Parkinson disease.

Although, by age 40, individuals with Down syndrome (DS) develop amyloid-β (Aβ) plaques and tau-containing neurofibrillary tangles (NFTs) linked to cognitive impairment in Alzheimer's disease (AD), not all people with DS develop dementia. Whether Aβ plaques and NFTs are associated with individuals with DS with (DSD +) and without dementia (DSD -) is under-investigated. Here, we applied quantitative immunocytochemistry and fluorescent procedures to characterize NFT pathology using antibodies specific for tau phosphorylation (pS422, AT8), truncation (TauC3, MN423), and conformational (Alz50, MC1) epitopes, as well as Aβ and its precursor protein (APP) to frontal cortex (FC) and striatal tissue from DSD + to DSD - cases. Expression profiling of single pS422 labeled FC layer V and VI neurons was also determined using laser capture microdissection and custom-designed microarray analysis. Analysis revealed that cortical and striatal Aβ plaque burdens were similar in DSD + and DSD - cases. In both groups, most FC plaques were neuritic, while striatal plaques were diffuse. By contrast, FC AT8-positive NFTs and neuropil thread densities were significantly greater in DSD + compared to DSD -, while striatal NFT densities were similar between groups. FC pS422-positive and TauC3 NFT densities were significantly greater than Alz50-labeled NFTs in DSD + , but not DSD - cases. Putaminal, but not caudate pS422-positive NFT density, was significantly greater than TauC3-positive NFTs. In the FC, AT8 + pS422 + Alz50, TauC3 + pS422 + Alz50, pS422 + Alz50, and TauC3 + pS422 positive NFTs were more frequent in DSD + compared to DSD- cases. Single gene-array profiling of FC pS422 positive neurons revealed downregulation of 63 of a total of 864 transcripts related to Aβ/tau biology, glutamatergic, cholinergic, and monoaminergic metabolism, intracellular signaling, cell homeostasis, and cell death in DSD + compared DSD - cases. These observations suggest that abnormal tau aggregation plays a critical role in the development of dementia in DS.

Lysosomes, single-membrane organelles defined by a uniquely acidic lumenal pH and high content of acid hydrolases, are the shared degradative compartments of the endocytic and autophagic pathways. These pathways, and especially lysosomes, are points of particular vulnerability in many neurodegenerative diseases. Beyond the role of lysosomes in substrate degradation, new findings have ascribed lysosomes with the leading role in sensing and responding to cellular nutrients, growth factors and cellular stress. This review aims to integrate recent concepts of basic lysosome biology and pathobiology as a basis for understanding neurodegenerative disease pathogenesis. Here, we discuss the newly recognized signaling functions of lysosomes and specific aspects of lysosome biology in neurons while re-visiting the classical defining criteria for lysosomes and the importance of strict definitions. Our discussion emphasizes dynein-mediated axonal transport of maturing degradative organelles, with further consideration of their roles in synaptic function. We finally examine how distinctive underlying disturbances of lysosomes in various neurodegenerative diseases result in unique patterns of auto/endolysosomal mistrafficking. The rapidly emerging understanding of lysosomal trafficking and disruptions in lysosome signaling is providing valuable clues to new targets in disease-modifying therapies.

In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the ɛ4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-β and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral ɛ3 allele we show that the ɛ4 allele, whether homozygous or heterozygous with an ɛ3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-β precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.

The dentate gyrus (DG) and its primary cell type, the granule cell (GC), are thought to be critical to many cognitive functions. A major neuronal subtype of the DG is the hilar mossy cell (MC). MCs have been considered to play an important role in cognition, but in vivo studies to understand the activity of MCs during cognitive tasks are challenging because the experiments usually involve trauma to the overlying hippocampus or DG, which kills hilar neurons. In addition, restraint typically occurs, and MC activity is reduced by brief restraint stress. Social isolation often occurs and is potentially confounding. Therefore, we used c-fos protein expression to understand when MCs are active in vivo in socially housed adult C57BL/6 mice in their home cage. We focused on c-fos protein expression after animals explored novel objects, based on previous work which showed that MCs express c-fos protein readily in response to a novel housing location. Also, MCs are required for the training component of the novel object location task and novelty-encoding during a food-related task. GluR2/3 was used as a marker of MCs. The results showed that MC c-fos protein is greatly increased after exposure to novel objects, especially in ventral DG. We also found that novel objects produced higher c-fos levels than familiar objects. Interestingly, a small subset of neurons that did not express GluR2/3 also increased c-fos protein after novel object exposure. In contrast, GCs appeared relatively insensitive. The results support a growing appreciation of the role of the DG in novelty detection and novel object recognition, where hilar neurons and especially MCs are very sensitive.