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Cell type specific enhancers for primate prefrontal cortex

ABSTRACT: Humans and nonhuman primates (NHPs) can reason, deliberate, and make causal inferences. It is now possible to study the cell types and circuits underlying these complex neural functions by using adeno-associated viral (AAV) vectors to deliver circuit-breaking tools that are driven by cell type-specific enhancers. Here, we set out to identify cell type specific enhancers for principal neurons in the dorsolateral prefrontal cortex. We used single nucleus RNA-Seq to define molecular phenotypes, and we used single nucleus ATAC-Seq to identify cell type specific open chromatin regions (OCRs). We used machine learning models that learned the regulatory vocabulary for each cell type and ranked 500nt OCRs according to their potential to drive cell type specific transgene expression – we call these 500nt sequences ‘enhancer candidates’. The top-ranked enhancer candidates for layer 3 pyramidal neurons (L3PNs) and a layer 5 pyramidal neuron extra-telencephalic (ET) subtype were packaged into AAVs with unique DNA barcodes. We use a combination of enhancer screening and one at a time validation to identify enhancers capable of driving cell type specific gene expression. The best performing ‘layer 3’ enhancer consistently restricted expression to layers 2 and 3. The performance of best performing layer 5 ET vector was titer dependent, but it demonstrates high specificity for POU3F1+ PN. This work provides vectors for examining cell types related to NHP cognitive functions and establishes a platform for future development of more cell type and circuit-specific AAVs that function in NHP brains.

ORGANISM(S): Macaca mulatta

PROVIDER: GSE279299 | GEO | 2025/05/22

REPOSITORIES: GEO

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Project description:Gray matter volume in the cerebral cortex has been consistently found to be decreased in patients with schizophrenia. The superior temporal gyrus (STG) is one of the cortical regions that exhibit the most pronounced volumetric reduction. This reduction is generally thought to reflect, at least in part, decreased number of synapses; the majority of these synapses are believed to be furnished by glutamatergic axon terminals onto the dendritic spines on pyramidal neurons. Pyramidal neurons in the cerebral cortex exhibit layer-specific connectional properties, providing neural circuit structures that support distinct aspects of higher cortical functions. For instance, dendritic spines on pyramidal neurons in layer 3 of the cerebral cortex are targeted by both local and long-range glutamatergic projections in a highly reciprocal fashion. Synchronized activities of pyramidal neuronal networks, especially in the gamma frequency band (i.e. 30-100 Hz), are critical for the integrity of higher cortical functions. Disturbances of these networks may contribute to the pathophysiology of schizophrenia by compromising gamma oscillation. This concept is supported by the following postmortem and clinical observations. First, the density of dendritic spines on pyramidal neurons in layer 3 of the cerebral cortex, including the STG, have been shown to be significantly decreased by 23-66% in subjects with schizophrenia. Second, consistent with these findings, the average somal area of these pyramidal cells is significantly smaller. Third, we have recently found that, in the prefrontal cortex, the density of glutamatergic axonal boutons, of which dendritic spines are their major targets, was significantly decreased by as much as 79% in layer 3 (but not layer 5) in subjects with schizophrenia. Finally, an increasing number of clinical studies have consistently demonstrated that gamma oscillatory synchrony is profoundly impaired in patients with schizophrenia. Furthermore, gamma impairment has been linked to the symptoms and cognitive deficits of the illness and the severity of these symptoms and deficits have in turn been associated with the magnitude of cortical gray matter reduction. Taken together, understanding the molecular underpinnings of pyramidal cell dysfunction will shed important light onto the pathophysiology of cortical dysfunction of schizophrenia. In order to gain insight into the molecular determinants of pyramidal cell dysfunction in schizophrenia, we combined LCM with Affymetrix microarray and high-throughput TaqManM-BM-.-based MegaPlex qRT-PCR approaches, respectively, to elucidate the alterations in messenger ribonucleic acid (mRNA) and microRNA (miRNA) expression profiles of these neurons in layer 3 of the STG. We found that transforming growth factor beta (TGFM-NM-2) and BMP (bone morphogenetic proteins) signaling pathways and many genes that regulate extracellular matrix (ECM), apoptosis and cytoskeleton were dysregulated in schizophrenia. In addition, we identified 10 miRNAs that were differentially expressed in this illness; interestingly, the predicted targets of these miRNAs included the dysregulated pathways and gene networks identified by microarray analysis. Together these findings provide a neurobiological framework within which we can begin to formulate and test specific hypotheses about the molecular mechanisms that underlie pyramidal cell dysfunction in schizophrenia. Gene epxression microarray from RNA isolated from pyramidal cells in layer III of the STG from 9 normal controls and 9 subjects with schizophrenia. There was no significant difference between diagnosis groups for age, sex, and post mortem interval (PMI).

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Cell type specific enhancers for primate prefrontal cortex

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