Analysis of microisolated frontal cortex excitatory layer III and V pyramidal neurons reveals a neurodegenerative phenotype in individuals with Down syndrome
Down syndrome (DS) is the most prevalent genetic abnormality, occurring in ~1/700 live births caused by triplication of human chromosome 21 (HSA21). DS individuals have a multitude of phenotypic changes in peripheral systems and most notably, are cognitively impaired, with deficits in learning, language and memory acquisition and consolidation. Further, individuals with DS develop Alzheimer's disease (AD) pathology during early middle-age (mid-30's), although the underlying mechanism(s) driving this pathology onset are not well understood. Contributing to cognitive impairment in DS, morphological deficits are present in cortical lamination and distribution, with Layer III (L3) and Layer V (L5) pyramidal neurons showing reduced proliferation and a disorganized laminar structure. As such, examining cortical L3 and L5 pyramidal neurons in DS individuals with AD pathology may elucidate alternate driver mechanisms of disease onset. We examined DS and age-matched control (CTR) brains from individuals without intellectual disabilities or dementia using postmortem frontal cortex (BA9) via single population low input RNA-sequencing. We specifically targeted L3 and L5 pyramidal neurons to understand circuitry-based alterations in DS/AD individuals to elucidate mechanistic drivers of disease pathology. We show convergent and divergent gene expression changes in L3 and L5 pyramidal neurons which may underlie cognitive deficits and degenerative pathology associated with aging in the DS brain. We pinpoint alterations in HSA21 gene expression as well as a multitude of convergent differentially expressed genes (DEGs) and relevant pathways via bioinformatic inquiry in the DS brains that underlie mechanisms of degeneration. Distinctly, convergent gene expression reveals a neurotoxic phenotype, with multiple canonical pathways of dysregulation and disease functions linked to the convergent DEGs, including downregulation of the CLEAR signaling pathway and significantly increased activation of the neuroinflammation signaling pathway. We identify several target genes including mitogen activated protein kinase 1 and 3 (MAPK1/3), calcium voltage-gated channel subunit alpha1 A (CACNA1A) and superoxide dismutase 1 (SOD1), which exhibit overlap in multiple pathways and disease functions and/or display dysfunctional protein-protein network interactions for further examination. Novel targets identified by our single population approach in L3 and L5 pyramidal neurons may be drivers of the disease mechanism and therefore may be therapeutic candidates for amelioration of degenerative pathology that targets memory and executive function circuits in DS/AD. Overall design: To determine gene expression changes in L3 and L5 pyramidal neurons from Down Syndrome compared to control brains, we isolated via LCM and purified RNA from single populations of pyramidal neurons spatially and morphologically identified before LCM.  RNA was purified from ~500 neurons for single population assays of L3 and L5 individual populations from the each brain specimen.  A total of n=12 DS (6M/6F) and n= 19 CTR (9M/8F) brains were captured via LCM for RNA-sequencing analysis of differentially expressed genes in age (42-67 yrs old), sex  and PMI matched cohorts. We purified RNA from LCM captured neurons.  RNA was split to perform library preparation on two techical replicates per layer per brain.  Sequencing was performed using New York University's Genome Technology Center with Illimina's NovaSeq 6000 platform. We compare the DS brains compared to controls in to determine gene expression differences due to genotype within each specific cortical layer.  We further analyzed the data to determine convergent and divergent gene expression between L3 and L5 pyramidal neurons due to the DS phenotype, to examine circuitry differences and overall alterations in cortical pyramidal neuron gene expression due to disease in aged samples (age range 42-67 yrs old).