Faculty Bibliography

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.

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.

Background: The brain contains interconnected circuits which are neither completed at birth or invariant across life. This neuronal plasticity is essential for life-long adaptive features like continuous learning and memory. However, this plasticity, especially when associated with severe adverse factors during early-life, can lead to the derailment of normative brain development and contribute to the etiology of behavioral deficits and psychiatric disorders. One of the most influential environmental factors during early-life is parental/caregiver care. Childhood adversity has been estimated to account for a significant percentage of adult-onset mental health disorders. In extreme cases of childhood adversity, institutional rearing where the infants were deprived of caregiver contact, cognitive deficits and dysregulated prefrontal cortex (PFC) function were found. In this study we hypothesize that earlylife adversity, in the form of maternal separation, leads to longlasting changes in the transcriptome of specific PFC cell populations leading to a dysregulation of PFC function.
Method(s): Here, we use a mouse model of maternal separation. The brains of pups and adults of normal reared or maternal separated (MS) animals were collected. In one set, we performed single-nucleus RNAseq with hashing to contrast the PFC transcriptome of these groups. In a separate cohort, we recorded whole slice voltage dye (VSD) responses to explore how changes in receptors identified by snRNAseq altered PFC responses.
Result(s): Using snRNAseq, as expected, we observed that the changes between ages were much more accentuated than changes between treatments. We also observed that the proportion of oligodendrocytes in adult-MS animals was similar to infant animals. Furthermore, we found that most of the differential gene expression between MS and standard reared animals was in interneurons, affecting pathways related to GABAergic, glutamatergic, and serotonergic functions. Using VSD we observed that adult-MS responses to GABAergic and serotonergic agonists were similar to the responses of more immature normal-reared animals.
Conclusion(s): This study suggests that MS leads to an immature PFC that may be linked to the behavioral deficits observed in animals and humans exposed to poor early-life care

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 β-cleaved soluble APP fragments (sAPPs) were similar across genotypes, as were the levels of the membrane-associated α- and β-cleaved C-terminal fragments (CTFs) of APP. Lastly, APOE genotype did not impact the level of soluble amyloid beta (Aβ). These findings argue that the APOE-allele-dependent AD risk is independent of the brain expression and processing of APP.

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.

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.

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.

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.

Since ancient times, tissue fixation for neurohistology has been an evolving area of research. Alcohol fixation first made possible examination of brain tissue specimens. In the late 1800s formaldehyde was introduced as a cross-linking fixative, which enabled histological advances. Various aldehyde solutions remain the staple for neurohistology in the neuroscience community including formalin, paraformaldehyde, glutaraldehyde, and combinations of these fixatives. A 4% paraformaldehyde solution is commonly used for numerous cytochemical procedures including tract tracing, immunohistochemistry, and in situ hybridization. A glutaraldehyde-paraformaldehyde solution is the fixative of choice for ultrastructural investigations using the electron microscope. With the advent of modern molecular and cellular biological techniques, it is now possible to isolate and study genomic DNA, RNA species, and proteins from microdissected tissue sources. Similar to light and electron microscopy, alcohol and aldehyde tissue fixation are compatible with preserving RNA integrity for regional and single cell gene array experiments using immersion- and perfusion-fixed tissue from a myriad of brain tissue sources including humans and relevant animal and cellular models.
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