Early-life prefrontal cortex inhibition and early-life stress lead to long-lasting behavioral, transcriptional, and physiological impairments.

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.

Maternal choline supplementation protects against age-associated cholinergic and GABAergic basal forebrain neuron degeneration in the Ts65Dn mouse model of Down syndrome and Alzheimer's disease.

Down syndrome (DS) is a genetic disorder caused by triplication of human chromosome 21. In addition to intellectual disability, DS is defined by a premature aging phenotype and Alzheimer's disease (AD) neuropathology, including septohippocampal circuit vulnerability and degeneration of basal forebrain cholinergic neurons (BFCNs). The Ts65Dn mouse model recapitulates key aspects of DS/AD pathology, namely age-associated atrophy of BFCNs and cognitive decline in septohippocampal-dependent behavioral tasks. We investigated whether maternal choline supplementation (MCS), a well-tolerated treatment modality, protects vulnerable BFCNs from age- and genotype-associated degeneration in trisomic offspring. We also examined the effect of trisomy, and MCS, on GABAergic basal forebrain parvalbumin neurons (BFPNs), an unexplored neuronal population in this DS model. Unbiased stereological analyses of choline acetyltransferase (ChAT)-immunoreactive BFCNs and parvalbumin-immunoreactive BFPNs were conducted using confocal z-stacks of the medial septal nucleus and the vertical limb of the diagonal band (MSN/VDB) in Ts65Dn mice and disomic (2N) littermates at 3-4 and 10-12 months of age. MCS trisomic offspring displayed significant increases in ChAT-immunoreactive neuron number and density compared to unsupplemented counterparts, as well as increases in the area of the MSN/VDB occupied by ChAT-immunoreactive neuropil. MCS also rescued BFPN number and density in Ts65Dn offspring, a novel rescue of a non-cholinergic cell population. Furthermore, MCS prevented age-associated loss of BFCNs and MSN/VDB regional area in 2N offspring, indicating genotype-independent neuroprotective benefits. These findings demonstrate MCS provides neuroprotection of vulnerable BFCNs and non-cholinergic septohippocampal BFPNs, indicating this modality has translational value as an early life therapy for DS, as well as extending benefits to the aging population at large.

Basal forebrain cholinergic neurons are vulnerable in a mouse model of Down syndrome and their molecular fingerprint is rescued by maternal choline supplementation.

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics in these disorders have been unsuccessful in slowing disease progression, likely due to poorly understood complex pathological interactions and dysregulated pathways. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration and has shown lifelong behavioral changes due to maternal choline supplementation (MCS). To test the impact of MCS on trisomic BFCNs, we performed laser capture microdissection to individually isolate choline acetyltransferase-immunopositive neurons in Ts65Dn and disomic littermates, in conjunction with MCS at the onset of BFCN degeneration. We utilized single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCNs. Leveraging multiple bioinformatic analysis programs on differentially expressed genes (DEGs) by genotype and diet, we identified key canonical pathways and altered physiological functions within Ts65Dn MSN BFCNs, which were attenuated by MCS in trisomic offspring, including the cholinergic, glutamatergic and GABAergic pathways. We linked differential gene expression bioinformatically to multiple neurological functions, including motor dysfunction/movement disorder, early onset neurological disease, ataxia and cognitive impairment via Ingenuity Pathway Analysis. DEGs within these identified pathways may underlie aberrant behavior in the DS mice, with MCS attenuating the underlying gene expression changes. We propose MCS ameliorates aberrant BFCN gene expression within the septohippocampal circuit of trisomic mice through normalization of principally the cholinergic, glutamatergic, and GABAergic signaling pathways, resulting in attenuation of underlying neurological disease functions.

Microisolation of Spatially Characterized Single Populations of Neurons for RNA Sequencing from Mouse and Postmortem Human Brain Tissues.

Single-cell and single-population RNA sequencing (RNA-seq) is a rapidly evolving new field of intense investigation. Recent studies indicate unique transcriptomic profiles are derived based on the spatial localization of neurons within circuits and regions. Individual neuronal subtypes can have vastly different transcriptomic fingerprints, well beyond the basic excitatory neuron and inhibitory neuron designations. To study single-population gene expression profiles of spatially characterized neurons, we have developed a methodology combining laser capture microdissection (LCM), RNA purification of single populations of neurons, and subsequent library preparation for downstream applications, including RNA-seq. LCM provides the benefit of isolating single neurons characterized by morphology or via transmitter-identified and/or receptor immunoreactivity and enables spatial localization within the sample. We utilize unfixed human postmortem and mouse brain tissue that is frozen to preserve RNA quality in order to isolate the desired neurons of interest. Microisolated neurons are then pooled for RNA purification utilizing as few as 250 individual neurons from a tissue section, precluding extraneous nonspecific tissue contaminants. Library preparation is performed from picogram RNA quantities extracted from LCM-captured neurons. Single-population RNA-seq analysis demonstrates that microisolated neurons from both postmortem human and mouse brain tissues are viable for transcriptomic profiling, including differential gene expression assessment and bioinformatic pathway inquiry.

Application of robust regression in translational neuroscience studies with non-Gaussian outcome data.

Linear regression is one of the most used statistical techniques in neuroscience, including the study of the neuropathology of Alzheimer's disease (AD) dementia. However, the practical utility of this approach is often limited because dependent variables are often highly skewed and fail to meet the assumption of normality. Applying linear regression analyses to highly skewed datasets can generate imprecise results, which lead to erroneous estimates derived from statistical models. Furthermore, the presence of outliers can introduce unwanted bias, which affect estimates derived from linear regression models. Although a variety of data transformations can be utilized to mitigate these problems, these approaches are also associated with various caveats. By contrast, a robust regression approach does not impose distributional assumptions on data allowing for results to be interpreted in a similar manner to that derived using a linear regression analysis. Here, we demonstrate the utility of applying robust regression to the analysis of data derived from studies of human brain neurodegeneration where the error distribution of a dependent variable does not meet the assumption of normality. We show that the application of a robust regression approach to two independent published human clinical neuropathologic data sets provides reliable estimates of associations. We also demonstrate that results from a linear regression analysis can be biased if the dependent variable is significantly skewed, further indicating robust regression as a suitable alternate approach.

Expression and proteolytic processing of the amyloid precursor protein is unaffected by the expression of the three human apolipoprotein E alleles in the brains of mice.

The 3 human apolipoprotein E (APOE) gene alleles modify an individual's risk of developing Alzheimer's disease (AD): compared to the risk-neutral APOE ε3 allele, the ε4 allele (APOE4) is strongly associated with increased AD risk while the ε2 allele is protective. Multiple mechanisms have been shown to link APOE4 expression and AD risk, including the possibility that APOE4 increases the expression of the amyloid precursor protein (APP) (Y-W.A. Huang, B. Zhou, A.M. Nabet, M. Wernig, T.C. Südhof, 2019). In this study, we investigated the impact of APOE genotype on the expression, and proteolytic processing of endogenously expressed APP in the brains of mice humanized for the 3 APOE alleles. In contrast to prior studies using neuronal cultures, we found in the brain that both App gene expression, and the levels of APP holoprotein were not affected by APOE genotype. Additionally, our analysis of APP fragments showed that APOE genotype does not impact APP processing in the brain: the levels of both α- and ß-cleaved soluble APP fragments (sAPPs) were similar across genotypes, as were the levels of the membrane-associated α- and ß-cleaved C-terminal fragments (CTFs) of APP. Lastly, APOE genotype did not impact the level of soluble amyloid beta (Aß). These findings argue that the APOE-allele-dependent AD risk is independent of the brain expression and processing of APP.

Aging in Down Syndrome: Latest Clinical Advances and Prospects.

Down syndrome (DS), or trisomy 21, is the most common genetic cause of intellectual disability [...].

Aging with Down Syndrome-Where Are We Now and Where Are We Going?

Down syndrome (DS) is a form of accelerated aging, and people with DS are highly prone to aging-related conditions that include vascular and neurological disorders. Due to the overexpression of several genes on Chromosome 21, for example genes encoding amyloid precursor protein (APP), superoxide dismutase (SOD), and some of the interferon receptors, those with DS exhibit significant accumulation of amyloid, phospho-tau, oxidative stress, neuronal loss, and neuroinflammation in the brain as they age. In this review, we will summarize the major strides in this research field that have been made in the last few decades, as well as discuss where we are now, and which research areas are considered essential for the field in the future. We examine the scientific history of DS bridging these milestones in research to current efforts in the field. We extrapolate on comorbidities associated with this phenotype and highlight clinical networks in the USA and Europe pursuing clinical research, concluding with funding efforts and recent recommendations to the NIH regarding DS research.

Oxidative Phosphorylation Is Dysregulated Within the Basocortical Circuit in a 6-month old Mouse Model of Down Syndrome and Alzheimer's Disease.

Down syndrome (DS) is the primary genetic cause of intellectual disability (ID), which is due to the triplication of human chromosome 21 (HSA21). In addition to ID, HSA21 trisomy results in a number of neurological and physiological pathologies in individuals with DS, including progressive cognitive dysfunction and learning and memory deficits which worsen with age. Further exacerbating neurological dysfunction associated with DS is the concomitant basal forebrain cholinergic neuron (BFCN) degeneration and onset of Alzheimer's disease (AD) pathology in early mid-life. Recent single population RNA sequencing (RNA-seq) analysis in the Ts65Dn mouse model of DS, specifically the medial septal cholinergic neurons of the basal forebrain (BF), revealed the mitochondrial oxidative phosphorylation pathway was significantly impacted, with a large subset of genes within this pathway being downregulated. We further queried oxidative phosphorylation pathway dysregulation in Ts65Dn mice by examining genes and encoded proteins within brain regions comprising the basocortical system at the start of BFCN degeneration (6 months of age). In select Ts65Dn mice we demonstrate significant deficits in gene and/or encoded protein levels of Complex I-V of the mitochondrial oxidative phosphorylation pathway in the BF. In the frontal cortex (Fr Ctx) these complexes had concomitant alterations in select gene expression but not of the proteins queried from Complex I-V, suggesting that defects at this time point in the BF are more severe and occur prior to cortical dysfunction within the basocortical circuit. We propose dysregulation within mitochondrial oxidative phosphorylation complexes is an early marker of cognitive decline onset and specifically linked to BFCN degeneration that may propagate pathology throughout cortical memory and executive function circuits in DS and AD.

Profiling Basal Forebrain Cholinergic Neurons Reveals a Molecular Basis for Vulnerability Within the Ts65Dn Model of Down Syndrome and Alzheimer's Disease.

Basal forebrain cholinergic neuron (BFCN) degeneration is a hallmark of Down syndrome (DS) and Alzheimer's disease (AD). Current therapeutics have been unsuccessful in slowing disease progression, likely due to complex pathological interactions and dysregulated pathways that are poorly understood. The Ts65Dn trisomic mouse model recapitulates both cognitive and morphological deficits of DS and AD, including BFCN degeneration. We utilized Ts65Dn mice to understand mechanisms underlying BFCN degeneration to identify novel targets for therapeutic intervention. We performed high-throughput, single population RNA sequencing (RNA-seq) to interrogate transcriptomic changes within medial septal nucleus (MSN) BFCNs, using laser capture microdissection to individually isolate ~500 choline acetyltransferase-immunopositive neurons in Ts65Dn and normal disomic (2N) mice at 6 months of age (MO). Ts65Dn mice had unique MSN BFCN transcriptomic profiles at ~6 MO clearly differentiating them from 2N mice. Leveraging Ingenuity Pathway Analysis and KEGG analysis, we linked differentially expressed gene (DEG) changes within MSN BFCNs to several canonical pathways and aberrant physiological functions. The dysregulated transcriptomic profile of trisomic BFCNs provides key information underscoring selective vulnerability within the septohippocampal circuit. We propose both expected and novel therapeutic targets for DS and AD, including specific DEGs within cholinergic, glutamatergic, GABAergic, and neurotrophin pathways, as well as select targets for repairing oxidative phosphorylation status in neurons. We demonstrate and validate this interrogative quantitative bioinformatic analysis of a key dysregulated neuronal population linking single population transcript changes to an established pathological hallmark associated with cognitive decline for therapeutic development in human DS and AD.

Adiponectin Modulation by Genotype and Maternal Choline Supplementation in a Mouse Model of Down Syndrome and Alzheimer's Disease.

Down syndrome (DS) is a genetic disorder caused by the triplication of human chromosome 21, which results in neurological and physiological pathologies. These deficits increase during aging and are exacerbated by cognitive decline and increase of Alzheimer's disease (AD) neuropathology. A nontoxic, noninvasive treatment, maternal choline supplementation (MCS) attenuates cognitive decline in mouse models of DS and AD. To evaluate potential underlying mechanisms, laser capture microdissection of individual neuronal populations of MCS offspring was performed, followed by RNA sequencing and bioinformatic inquiry. Results at ~6 months of age (MO) revealed DS mice (the well-established Ts65Dn model) have significant dysregulation of select genes within the Type 2 Diabetes Mellitus (T2DM) signaling pathway relative to normal disomic (2N) littermates. Accordingly, we interrogated key T2DM protein hormones by ELISA assay in addition to gene and encoded protein levels in the brain. We found dysregulation of adiponectin (APN) protein levels in the frontal cortex of ~6 MO trisomic mice, which was attenuated by MCS. APN receptors also displayed expression level changes in response to MCS. APN is a potential biomarker for AD pathology and may be relevant in DS. We posit that changes in APN signaling may be an early marker of cognitive decline and neurodegeneration.

Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome.

Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.

Brain-derived neurotrophic factor (BDNF) and TrkB hippocampal gene expression are putative predictors of neuritic plaque and neurofibrillary tangle pathology.

INTRODUCTION: Downregulation of brain-derived neurotrophic factor (BDNF) and its cognate neurotrophin receptor, TrkB, were observed during the progression of dementia, but whether the Alzheimer's disease (AD) pathological lesions diffuse plaques, (DPs), neuritic plaques (NPs), and neurofibrillary tangles (NFTs) are related to this alteration remains to be clarified. METHODS: Negative binomial (NB) regressions were performed using gene expression data accrued from a single population of CA1 pyramidal neurons and regional hippocampal dissections obtained from participants in the Rush Religious Orders Study (RROS). RESULTS: Downregulation of Bdnf is independently associated with increased entorhinal cortex NPs. Downregulation of TrkB is independently associated with increased entorhinal cortex NFTs and CA1 NPs during the progression of AD. DISCUSSION: Results indicate that BDNF and TrkB dysregulation contribute to AD neuropathology, most notably hippocampal NPs and NFTs. These data suggest attenuating BDNF/TrkB signaling deficits either at the level of BDNF, TrkB, or downstream of TrkB signaling may abrogate NPs and/or NFTs.

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.

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.

Maternal Choline Supplementation Alters Basal Forebrain Cholinergic Neuron Gene Expression in the Ts65Dn Mouse Model of Down Syndrome.

Down syndrome (DS), trisomy 21, is marked by intellectual disability and a premature aging profile including degeneration of the basal forebrain cholinergic neuron (BFCN) projection system, similar to Alzheimer's disease (AD). Although data indicate that perinatal maternal choline supplementation (MCS) alters the structure and function of these neurons in the Ts65Dn mouse model of DS and AD (Ts), whether MCS affects the molecular profile of vulnerable BFCNs remains unknown. We investigated the genetic signature of BFCNs obtained from Ts and disomic (2N) offspring of Ts65Dn dams maintained on a MCS diet (Ts+, 2N+) or a choline normal diet (ND) from mating until weaning, then maintained on ND until 4.4-7.5 months of age. Brains were then collected and prepared for choline acetyltransferase (ChAT) immunohistochemistry and laser capture microdissection followed by RNA extraction and custom-designed microarray analysis. Findings revealed upregulation of select transcripts in classes of genes related to the cytoskeleton (Tubb4b), AD (Cav1), cell death (Bcl2), presynaptic (Syngr1), immediate early (Fosb, Arc), G protein signaling (Gabarap, Rgs10), and cholinergic neurotransmission (Chrnb3) in Ts compared to 2N mice, which were normalized with MCS. Moreover, significant downregulation was seen in select transcripts associated with the cytoskeleton (Dync1h1), intracellular signaling (Itpka, Gng3, and Mlst8), and cell death (Ccng1) in Ts compared to 2N mice that was normalized with MCS. This study provides insight into genotype-dependent differences and the effects of MCS at the molecular level within a key vulnerable cell type in DS and AD.

Frontal cortex and striatal cellular and molecular pathobiology in individuals with Down syndrome with and without dementia.

Although, by age 40, individuals with Down syndrome (DS) develop amyloid-ß (Aß) plaques and tau-containing neurofibrillary tangles (NFTs) linked to cognitive impairment in Alzheimer's disease (AD), not all people with DS develop dementia. Whether Aß 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 Aß 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 Aß 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 Aß/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.

Selective decline of neurotrophin and neurotrophin receptor genes within CA1 pyramidal neurons and hippocampus proper: Correlation with cognitive performance and neuropathology in mild cognitive impairment and Alzheimer's disease.

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 hippocampus 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). These 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.

Apolipoprotein E4 genotype compromises brain exosome production.

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-ß 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-ß 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.

CA1 pyramidal neuron gene expression mosaics in the Ts65Dn murine model of Down syndrome and Alzheimer's disease following maternal choline supplementation.

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 therapeutic intervention, 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 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 (Aß) 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.