Faculty Bibliography

Down syndrome (DS), stemming from the triplication of human chromosome 21, results in intellectual disability, with early mid-life onset of Alzheimer's disease (AD) pathology. Early interventions to reduce cognitive impairments and neuropathology are lacking. One modality, maternal choline supplementation (MCS), has shown beneficial effects on behavior and gene expression in neurodevelopmental and neurodegenerative disorders, including trisomic mice. Loss of basal forebrain cholinergic neurons (BFCNs) and other DS/AD relevant hallmarks were observed in a well-established trisomic model (Ts65Dn, Ts). MCS attenuates these endophenotypes with beneficial behavioral effects in trisomic offspring. We postulate MCS ameliorates dysregulated cellular mechanisms within vulnerable BFCNs, with attenuation driven by novel gene expression. Here, choline acetyltransferase immunohistochemical labeling identified BFCNs in the medial septal/ventral diagonal band nuclei of the basal forebrain in Ts and normal disomic (2N) offspring at ~11 months of age from dams exposed to MCS or normal choline during the perinatal period. BFCNs (~500 per mouse) were microisolated and processed for RNA-sequencing. Bioinformatic assessment elucidated differentially expressed genes (DEGs) and pathway alterations in the context of genotype (Ts, 2N) and maternal diet (MCS, normal choline). MCS attenuated select dysregulated DEGs and relevant pathways in aged BFCNs. Trisomic MCS-responsive improvements included pathways such as cognitive impairment and nicotinamide adenine dinucleotide signaling, among others, indicative of increased behavioral and bioenergetic fitness. Although MCS does not eliminate the DS/AD phenotype, early choline delivery provides long-lasting benefits to aged trisomic BFCNs, indicating that MCS prolongs neuronal health in the context of DS/AD.

INTRODUCTION/BACKGROUND:Underlying causes of Alzheimer's disease (AD) remain unknown, making it imperative to identify molecular mechanisms driving the pathobiology of AD onset and progression. METHODS:Laser capture microdissection was used to isolate layer III pyramidal neurons from post mortem human prefrontal cortex (Brodmann area 9). Single population RNA sequencing was conducted using tissue from subjects with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD. Differentially expressed genes (DEGs) were compared across groups. RESULTS:DEGs increased from prodromal (MCI vs. NCI) to progression (AD vs. MCI) to frank AD (AD vs. NCI). The majority of DEGs and pathways shared between prodromal and progression exhibited a change in the direction of dysregulation unlike pathways between progression and frank AD. DISCUSSION/CONCLUSIONS:Candidate genes and pathways were identified that demarcate early-stage AD onset from AD progression, providing a roadmap to study cortical cellular vulnerability and key targets for intervention at early stages of AD. HIGHLIGHTS/CONCLUSIONS:Pyramidal neuron differentially expressed genes (DEGs) are directionally divergent between prodromal, progression, and frank Alzheimer's disease (AD). Pyramidal neuron DEGs are directionally convergent between progression and frank AD. Dysfunctional bioenergetic pathways increased dysregulation as the AD spectrum progressed. Immune response pathways were more dysregulated in frank AD than prodromal stages. DEGs, = biological pathways, and interactomes demarcate specific stages across the AD spectrum.

BACKGROUND:/calmodulin-dependent protein kinase II and selected for probe collection. RESULTS:This approach significantly reduced the amount of FFPE tissue needed for robust single population RNA-seq. We demonstrate ~20 identified L3 or L5 pyramidal neurons or one lamina-specific cortical ribbon from a single 5µm thick section is sufficient to generate robust RNA-seq reads. Bioinformatic analysis of neurons and ribbons showed notable similarities and differences reflective of the single neuron and multiple admixed cell types within the former and latter, respectively. Comparison with existing methods Protocols exist for DSP of postmortem human FFPE brain tissue. However, this new approach enables profiling small groups of ~14-21 pyramidal neurons using the GeoMx DSP platform. CONCLUSIONS:This optimized DSP assay provides high resolution RNA-seq data demonstrating utility and versatility of the GeoMx platform for individually characterized neurons and isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses.

INTRODUCTION/UNASSIGNED:Individuals with Down syndrome (DS) exhibit neurological deficits throughout life including the development of in Alzheimer's disease (AD) pathology and cognitive impairment. At the cellular level, dysregulation in neuronal gene expression is observed in postmortem human brain and mouse models of DS/AD. To date, RNA-sequencing (RNA-seq) analysis of hippocampal neuronal gene expression including the characterization of discrete circuit-based connectivity in DS remains a major knowledge gap. We postulate that spatially characterized hippocampal neurons display unique gene expression patterns due, in part, to dysfunction of the integrity of intrinsic circuitry. METHODS/UNASSIGNED:We combined laser capture microdissection to microisolate individual neuron populations with single population RNA-seq analysis to determine gene expression analysis of CA1 and CA3 pyramidal neurons and dentate gyrus granule cells located in the hippocampus, a region critical for learning, memory, and synaptic activity. RESULTS/UNASSIGNED:The hippocampus exhibits age-dependent neurodegeneration beginning at ~6 months of age in the Ts65Dn mouse model of DS/AD. Each population of excitatory hippocampal neurons exhibited unique gene expression alterations in Ts65Dn mice. Bioinformatic inquiry revealed unique vulnerabilities and differences with mechanistic implications coinciding with onset of degeneration in this model of DS/AD. CONCLUSIONS/UNASSIGNED:These cell-type specific vulnerabilities may underlie degenerative endophenotypes suggesting precision medicine targeting of individual populations of neurons for rational therapeutic development.

Selective vulnerability of neuronal populations occurs in both Down syndrome (DS) and Alzheimer's disease (AD), resulting in disproportional degeneration of pyramidal neurons (PNs) affecting memory and executive function. Elucidating the cellular mechanisms underlying the selective vulnerability of these populations will provide pivotal insights for disease progression in DS and AD. Single population RNA-sequencing analysis was performed on neurons critical for executive function, prefrontal cortex Brodmann area 9 (BA9) layer III (L3) and layer V (L5) excitatory PNs in postmortem human DS and age- and sex-matched control (CTR) brains. Data mining was performed on differentially expressed genes (DEGs) from PNs in each lamina with DEGs divergent between lamina identified and interrogated. Bioinformatic inquiry of L3 PNs revealed more unique/differentially expressed DEGs (uDEGs) than in L5 PNs in DS compared to CTR subjects, indicating gene dysregulation shows both spatial and cortical laminar projection neuron dependent dysregulation. DS triplicated human chromosome 21 (HSA21) comprised a subset of DEGs only dysregulated in L3 or L5 neurons, demonstrating partial cellular specificity in HSA21 expression. These HSA21 uDEGs had a disproportionally high number of noncoding RNAs, suggesting lamina specific dysfunctional gene regulation. L3 uDEGs revealed overall more dysregulation of cellular pathways and processes, many relevant to early AD pathogenesis, while L5 revealed processes suggestive of frank AD pathology. These findings indicate that trisomy differentially affects a subpopulation of uDEGs in L3 and L5 BA9 projection neurons in aged individuals with DS, which may inform circuit specific pathogenesis underlying DS and AD.

We elucidated the molecular fingerprint of vulnerable excitatory neurons within select cortical lamina of individuals with Down syndrome (DS) for mechanistic understanding and therapeutic potential that also informs Alzheimer's disease (AD) pathophysiology. Frontal cortex (BA9) layer III (L3) and layer V (L5) pyramidal neurons were microisolated from postmortem human DS and age- and sex-matched controls (CTR) to interrogate differentially expressed genes (DEGs) and key biological pathways relevant to neurodegenerative programs. We identified > 2300 DEGs exhibiting convergent dysregulation of gene expression in both L3 and L5 pyramidal neurons in individuals with DS versus CTR subjects. DEGs included over 100 triplicated human chromosome 21 genes in L3 and L5 neurons, demonstrating a trisomic neuronal karyotype in both laminae. In addition, thousands of other DEGs were identified, indicating gene dysregulation is not limited to trisomic genes in the aged DS brain, which we postulate is relevant to AD pathobiology. Convergent L3 and L5 DEGs highlighted pertinent biological pathways and identified key pathway-associated targets likely underlying corticocortical neurodegeneration and related cognitive decline in individuals with DS. Select key DEGs were interrogated as potential hub genes driving dysregulation, namely the triplicated DEGs amyloid precursor protein (APP) and superoxide dismutase 1 (SOD1), along with key signaling DEGs including mitogen activated protein kinase 1 and 3 (MAPK1, MAPK3) and calcium calmodulin dependent protein kinase II alpha (CAMK2A), among others. Hub DEGs determined from multiple pathway analyses identified potential therapeutic candidates for amelioration of cortical neuron dysfunction and cognitive decline in DS with translational relevance to AD.

Early-life stress has been linked to multiple neurodevelopmental and neuropsychiatric deficits. Our previous studies have linked maternal presence/absence from the nest in developing rat pups to changes in prefrontal cortex (PFC) activity. Furthermore, we have shown that these changes are modulated by serotonergic signaling. Here we test whether changes in PFC activity during early life affect the developing cortex leading to behavioral alterations in the adult. We show that inhibiting the PFC of mouse pups leads to cognitive deficits in the adult comparable to those seen following maternal separation. Moreover, we show that activating the PFC during maternal separation can prevent these behavioral deficits. To test how maternal separation affects the transcriptional profile of the PFC we performed single-nucleus RNA-sequencing. Maternal separation led to differential gene expression almost exclusively in inhibitory neurons. Among others, we found changes in GABAergic and serotonergic pathways in these interneurons. Interestingly, both maternal separation and early-life PFC inhibition led to changes in physiological responses in prefrontal activity to GABAergic and serotonergic antagonists that were similar to the responses of more immature brains. Prefrontal activation during maternal separation prevented these changes. These data point to a crucial role of PFC activity during early life in behavioral expression in adulthood.

Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.

BACKGROUND/UNASSIGNED:Individuals with Down syndrome (DS) have intellectual disability and develop Alzheimer's disease (AD) pathology during midlife, particularly in the hippocampal component of the medial temporal lobe memory circuit. However, molecular and cellular mechanisms underlying selective vulnerability of hippocampal CA1 neurons remains a major knowledge gap during DS/AD onset. This is compounded by evidence showing spatial (e.g., deep versus superficial) localization of pyramidal neurons (PNs) has profound effects on activity and innervation within the CA1 region. OBJECTIVE/UNASSIGNED:We investigated whether there is a spatial profiling difference in CA1 PNs in an aged female DS/AD mouse model. We posit dysfunction may be dependent on spatial localization and innervation patterns within discrete CA1 subfields. METHODS/UNASSIGNED:Laser capture microdissection was performed on trisomic CA1 PNs in an established mouse model of DS/AD compared to disomic controls, isolating the entire CA1 pyramidal neuron layer and sublayer microisolations of deep and superficial PNs from the distal CA1 (CA1a) region. RESULTS/UNASSIGNED:RNA sequencing and bioinformatic inquiry revealed dysregulation of CA1 PNs based on spatial location and innervation patterns. The entire CA1 region displayed the most differentially expressed genes (DEGs) in trisomic mice reflecting innate DS vulnerability, while trisomic CA1a deep PNs exhibited fewer but more physiologically relevant DEGs, as evidenced by bioinformatic inquiry. CONCLUSIONS/UNASSIGNED:CA1a deep neurons displayed numerous DEGs linked to cognitive functions whereas CA1a superficial neurons, with approximately equal numbers of DEGs, were not linked to pathways of dysregulation, suggesting the spatial location of vulnerable CA1 PNs plays an important role in circuit dissolution.