Faculty Bibliography

Cholinergic basal forebrain neurons of the nucleus basalis of Meynert (nbM) regulate attentional and memory function and are exquisitely prone to tau pathology and neurofibrillary tangle (NFT) formation during the progression of Alzheimer's disease (AD). nbM neurons require the neurotrophin nerve growth factor (NGF), its cognate receptor TrkA, and the pan-neurotrophin receptor p75^NTR for their maintenance and survival. Additionally, nbM neuronal activity and cholinergic tone are regulated by the expression of nicotinic (nAChR) and muscarinic (mAChR) acetylcholine receptors as well as receptors modulating glutamatergic and catecholaminergic afferent signaling. To date, the molecular and cellular relationships between the evolution of tau pathology and nbM neuronal survival remain unknown. To address this knowledge gap, we profiled cholinotrophic pathway genes within nbM neurons immunostained for pS422, a pretangle phosphorylation event preceding tau C-terminal truncation at D421, or dual-labeled for pS422 and TauC3, a later stage tau neo-epitope revealed by this same C-terminal truncation event, via single-population custom microarray analysis. nbM neurons were obtained from postmortem tissues from subjects who died with an antemortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. Quantitative analysis revealed significant downregulation of mRNAs encoding TrkA as well as TrkB and the Trk-mediated downstream pro-survival kinase Akt in pS422+ compared to unlabeled, pS422-negative nbM neurons. In addition, pS422+ neurons displayed a downregulation of transcripts encoding NMDA receptor subunit 2B, metabotropic glutamate receptor 2, D2 dopamine receptor, and β1 adrenoceptor. By contrast, transcripts encoding p75^NTR were downregulated in dual-labeled pS422+/TauC3+ neurons. Appearance of the TauC3 epitope was also associated with an upregulation of the α7 nAChR subunit and differential downregulation of the β2 nAChR subunit. Notably, we found that gene expression patterns for each cell phenotype did not differ with clinical diagnosis. However, linear regression revealed that global cognition and Braak stage were predictors of select transcript changes within both unlabeled and pS422+/TauC3- neurons. Taken together, these cell phenotype-specific gene expression profiling data suggest that dysregulation of neurotrophic and neurotransmitter signaling is an early pathogenic mechanism associated with NFT formation in vulnerable nbM neurons and cognitive decline in AD, which may be amenable to therapeutic intervention early in the disease process.

Military personnel and athletes exposed to traumatic brain injury may develop chronic traumatic encephalopathy (CTE). Brain pathology in CTE includes intracellular accumulation of abnormally phosphorylated tau proteins (p-tau), the main constituent of neurofibrillary tangles (NFTs). Recently, we found that cholinergic basal forebrain (CBF) neurons within the nucleus basalis of Meynert (nbM), which provide the major cholinergic innervation to the cortex, display an increasing number of NFTs across the pathological stages of CTE.1 However, molecular mechanisms underlying nbM neurodegeneration post CTE remain unknown. Here, we assessed the genetic signature of nbM neurons containing the p-tau pretangle maker pS422 obtained from CTE subjects who came to autopsy and received a neuropathological CTE staging assessment (Stages II, III, and IV) using laser capture microdissection and custom-designed microarray analysis. Quantitative analysis revealed dysregulation of key genes in several gene ontology groups between CTE stages. Specifically, downregulation of the nicotinic cholinergic receptor subunit beta-2 gene (Chrnb2), monoaminergic enzymes catechol-O-methyltransferase (Comt) and dopa decarboxylase (Ddc), chloride channels Clcn4 and Clcn5, scaffolding protein caveolin 1 (Cav1), cortical development/cytoskeleton element lissencephaly 1 (Lis1) and intracellular signaling cascade member adenylate cyclase 3 (Adcy3) was observed in pS422-immunreactive nbM neurons in CTE patients. By contrast, upregulation of calpain 2 (Capn2) and microtubule-associated protein 2 (Map2) transcript levels was found in stage IV CTE patients. These single-population data in vulnerable neurons indicates alterations in gene expression associated with neurotransmission, signal transduction, the cytoskeleton, cell survival/death signaling, and microtubule dynamics suggesting novel molecular pathways to target for drug discovery in CTE.

Although there are changes in gene expression and alterations in neuronal density and afferent inputs in the forebrain of trisomic mouse models of Down syndrome (DS) and Alzheimer's disease (AD), there is a lack of systematic assessments of gene expression and encoded proteins within individual vulnerable cell populations, precluding translational investigations at the molecular and cellular level. Further, no effective treatment exists to combat intellectual disability and basal forebrain cholinergic neurodegeneration seen in DS. To further our understanding of gene expression changes before and following cholinergic degeneration in a well-established mouse model of DS/AD, the Ts65Dn mouse, we assessed RNA expression levels from CA1 pyramidal neurons at two adult ages (∼6 months of age and ∼11 months of age) in both Ts65Dn and their normal disomic (2N) littermates. We further examined a viable therapeutic, maternal choline supplementation (MCS), which has been previously shown to lessen dysfunction in spatial cognition and attention, and have protective effects on the survival of basal forebrain cholinergic neurons (BFCNs) in the Ts65Dn mouse model. Results indicate that MCS normalized expression of several genes in key gene ontology categories, including synaptic plasticity, calcium signaling, and AD-associated neurodegeneration related to amyloid-beta peptide (Aβ) clearance. Specifically, normalized expression levels were found for endothelin converting enzyme-2 (Ece2), insulin degrading enzyme (Ide), Dyrk1a, and calcium/calmodulin-dependent protein kinase II (Camk2a), among other relevant genes. Single population expression profiling of vulnerable CA1 pyramidal neurons indicates that MCS is a viable therapeutic for long-term reprogramming of key transcripts involved in neuronal signaling that are dysregulated in the trisomic mouse brain which have translational potential for DS and AD.

The energetic costs of behavioral chronic stress are unlikely to be sustainable without neuronal plasticity. Mitochondria have the capacity to handle synaptic activity up to a limit before energetic depletion occurs. Protective mechanisms driven by the induction of neuronal genes likely evolved to buffer the consequences of chronic stress on excitatory neurons in prefrontal cortex (PFC), as this circuitry is vulnerable to excitotoxic insults. Little is known about the genes involved in mitochondrial adaptation to the build up of chronic stress. Using combinations of genetic manipulations and stress for analyzing structural, transcriptional, mitochondrial and behavioral outcomes, we characterized NR4A1 as a stress-inducible modifier of mitochondrial energetic competence and dendritic spine number in PFC. NR4A1 acted as transcription factor for changing the expression of target genes previously involved in mitochondrial uncoupling, AMPK activation and synaptic growth. Maintenance of NR4A1 activity by chronic stress played a critical role in the regressive synaptic organization in PFC of mouse models of stress (male only). Knockdown, dominant negative and knockout of NR4A1 in mice and rats (male only) protected pyramidal neurons against the adverse effects of chronic stress. In human PFC tissues of men and women, high levels of the transcriptionally-active NR4A1 correlated with measures of synaptic loss and cognitive impairment. In the context of chronic stress, prolonged expression and activity of NR4A1 may lead to responses of mitochondria and synaptic connectivity that do not match environmental demand, resulting in circuit malfunction between PFC and other brain regions constituting a pathological feature across disorders.SIGNIFICANCE STATEMENTThe bioenergetics cost of chronic stress is too high to be sustainable by pyramidal prefrontal neurons. Cellular checkpoints have evolved to adjust responses of mitochondria and synapses to the build up of chronic stress. NR4A1 plays such role by controlling mitochondria energetic competence with respect to synapse number. As an immediate-early gene, NR4A1 promotes neuronal plasticity but sustained expression or activity can be detrimental. NR4A1 expression and activity is sustained by chronic stress in animal models and in human studies of neuropathologies sensitive to the build up of chronic stress. Therefore, antagonism of NR4A1 is a promising avenue for preventing the regressive synaptic reorganization in cortical systems in the context of chronic stress.

OBJECTIVE:CD16+ /CD163+ macrophages (MΦ)s and microglia accumulate in the brains of patients with HIV encephalitis (HIVE), a neuropathological correlate of the most severe form of HIV-associated neurocognitive disorders (HAND), HIV-associated dementia (HIV-D). Recently, we found that some parenchymal microglia in brain of HIV+ subjects without encephalitis (HIV/noE) but with varying degrees of neurocognitive impairment express CD16 and CD163, even in the absence of detectable virus production. To further our understanding of microglial activation in HIV, we investigated expression of specific genes by profiling parenchymal microglia from archival brain tissue of patients with HIVE, HIV/noE, and HIV- controls. METHODS:Single-population microarray analyses were performed on ∼2,500 laser capture microdissected CD163+ , CD16+ or CD68+ MΦs/microglia per case, using terminal continuation (TC) RNA amplification and a custom-designed array platform. RESULTS:Several classes of microglial transcripts in HIVE and HIV/noE, were altered, relative to HIV- subjects, including factors related to cell stress, immune activation, and apoptosis. Additionally, several neurotrophic factors are reduced in HIV infection, suggesting an additional mechanism of neuropathogenesis. The majority of transcripts altered in HIVE displayed intermediate changes in HIV/noE. INTERPRETATION/CONCLUSIONS:Our results support the notion that microglia contribute to the maintenance of brain homeostasis and their potential loss of function in the context of chronic inflammation contributes to neuropathogenesis. Furthermore, they indicate the utility of profiling MΦs/microglia to increase our understanding of microglia function, as well as ascertain alterations in specific pathways, genes, and, ostensibly, encoded proteins that may be amenable to targeted treatment modalities in diseases affecting the brain.

Background: The apolipoprotein E (APOE) gene codes for the brain's primary cholesterol carrier protein. In both humans and humanized APOE mice the Alzheimer's disease-risk APOE 4 allele (APOE4) alters the number and size of neuronal endosomes, a pathology common to several neurodegenerative disorders, including Alzheimer's disease. Given that exosomes derive from the endosomal system, we investigated the impact of APOE4 on brain-derived exosomes. Methods: Extracellular vesicles (EV) were isolated from brain tissue of neuropathologically normal humans and of APOE targeted-replacement mice at 6, 12 and 18 months of age. Antibodies against TSG101 and ALIX were used to identify the exosome population within these samples. Protein, mRNA and lipid analyses were performed on both EV and whole-brain samples. Results: We found lower exosome levels in the brains of neuropathologically normal human APOE4 carriers compared to individuals homozygous for the risk-neutral 3 allele (APOE3). In APOE4 compared with APOE3 mice, brain exosome levels were lower in an age-dependent manner: lower levels were observed at 12 and 18 but not at 6 months of age. Protein and mRNA expressions of the exosome pathway regulators TSG101 and Rab35 were also lower in APOE4 compared with APOE3 mouse brains at 12 months of age, arguing for decreased exosome biosynthesis and secretion, respectively, from the endosomal pathway. Cholesterol and ganglioside levels were higher in brain exosomes isolated from 12-month-old APOE4 compared with APOE3 mice. Summary/Conclusion: Our findings show an APOE4-driven downregulation of brain exosome biosynthesis and release that is associated with altered lipid homeostasis. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging, which is essential for diverse homeostatic and catabolic cellular processes, is likely to contribute to neuronal vulnerability in neurodegenerative disorders, including Alzheimer's disease

A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.

A major feature of Alzheimer's disease (AD) is the loss of noradrenergic locus coeruleus (LC) projection neurons that mediate attention, memory, and arousal. However, the extent to which the LC projection system degenerates during the initial stages of AD is still under investigation. To address this question, we performed tyrosine hydroxylase (TH) immunohistochemistry and unbiased stereology of noradrenergic LC neurons in tissue harvested postmortem from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), amnestic mild cognitive impairment (aMCI, a putative prodromal AD stage), or mild/moderate AD. Stereologic estimates of total LC neuron number revealed a 30% loss during the transition from NCI to aMCI, with an additional 25% loss of LC neurons in AD. Decreases in noradrenergic LC neuron number were significantly associated with worsening antemortem global cognitive function as well as poorer performance on neuropsychological tests of episodic memory, semantic memory, working memory, perceptual speed, and visuospatial ability. Reduced LC neuron numbers were also associated with increased postmortem neuropathology. To examine the cellular and molecular pathogenic processes underlying LC neurodegeneration in aMCI, we performed single population microarray analysis. These studies revealed significant reductions in select functional classes of mRNAs regulating mitochondrial respiration, redox homeostasis, and neuritic structural plasticity in neurons accessed from both aMCI and AD subjects compared to NCI. Specific gene expression levels within these functional classes were also associated with global cognitive deterioration and neuropathological burden. Taken together, these observations suggest that noradrenergic LC cellular and molecular pathology is a prominent feature of prodromal disease that contributes to cognitive dysfunction. Moreover, they lend support to a rational basis for targeting LC neuroprotection as a disease modifying strategy.

The Ts65Dn mouse model of Down syndrome (DS) and Alzheimer's disease (AD) exhibits cognitive impairment and degeneration of basal forebrain cholinergic neurons (BFCNs). Our prior studies demonstrated that maternal choline supplementation (MCS) improves attention and spatial cognition in Ts65Dn offspring, normalizes hippocampal neurogenesis, and lessens BFCN degeneration in the medial septal nucleus (MSN). Here we determined whether (i) BFCN degeneration contributes to attentional dysfunction, and (ii) whether the attentional benefits of perinatal MCS are due to changes in BFCN morphology. Ts65Dn dams were fed either a choline-supplemented or standard diet during pregnancy and lactation. Ts65Dn and disomic (2N) control offspring were tested as adults (12-17months of age) on a series of operant attention tasks, followed by morphometric assessment of BFCNs. Ts65Dn mice demonstrated impaired learning and attention relative to 2N mice, and MCS significantly improved these functions in both genotypes. We also found, for the first time, that the number of BFCNs in the nucleus basalis of Meynert/substantia innominata (NMB/SI) was significantly increased in Ts65Dn mice relative to controls. In contrast, the number of BFCNs in the MSN was significantly decreased. Another novel finding was that the volume of BFCNs in both basal forebrain regions was significantly larger in Ts65Dn mice. MCS did not normalize any of these morphological abnormalities in the NBM/SI or MSN. Finally, correlational analysis revealed that attentional performance was inversely associated with BFCN volume, and positively associated with BFCN density. These results support the lifelong attentional benefits of MCS for Ts65Dn and 2N offspring and have profound implications for translation to human DS and pathology attenuation in AD.

Aims Glucocorticoid resistance is a risk factor for Alzheimer's disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. OBJECTIVES: To test (i) the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance, (ii) its influence on dendritic spine loss and tau phosphorylation as risk factors for AD, and (iii) its relevance for human pathology. Method We manipulated (1) BDNF signaling using a TrkB mutant that can be inactivated chemically, (2) glucocorticoid signaling using a BDNF insensitive GR mutant, and (3) the expression of DUSP1, the MAPK-phosphatase downstream of BDNF and GR pathways in a mouse model of glucocorticoid resistance featuring impaired cortisol awaking response. Synaptic defects and Tau phosphorylation were analyzed post-mortem. DUSP1 expression in human brain was analyzed in correlation to AD diagnosis and cognitive impairment in two independent American cohorts (10 controls + 15 AD and 17 controls + 29 AD). Results Deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. Conclusion Our findings provide evidence for a causal role of BDNF-dependent GR signaling on tau neuropathology and indicate that DUSP1 is potential target of therapeutics