Disentangling transcriptomic heterogeneity within the human subgenual anterior cingulate cortex.

The subgenual anterior cingulate cortex (sgACC) is a critical site for understanding the neural correlates of affect and emotion. While the activity of the sgACC is functionally homogenous, it is comprised of multiple Brodmann Areas (BAs) that possess different cytoarchitectures. In some sgACC BAs, Layer 5 is sublaminated into L5a and L5b which has implications for its projection targets. To understand how the transcriptional profile differs between the BAs, layers, and sublayers of human sgACC, we collected layer strips using laser capture microdissection followed by RNA sequencing. We found no significant differences in transcript expression in these specific cortical layers between BAs within the sgACC. In contrast, we identified striking differences between Layers 3 and 5a or 5b that were concordant across sgACC BAs. We found that sublayers 5a and 5b were transcriptionally similar. Pathway analyses of L3 and L5 revealed overlapping biological processes related to synaptic function. However, L3 was enriched for pathways related to cell-to-cell junction and dendritic spines whereas L5 was enriched for pathways related to brain development and presynaptic function, indicating potential functional differences across layers. Our study provides important insight into normative transcriptional features of the sgACC.

Characterization of the three-dimensional synaptic and mitochondrial nanoarchitecture within glutamatergic synaptic complexes in postmortem human brain via focused ion beam-scanning electron microscopy.

Glutamatergic synapses are the primary site of excitatory synaptic signaling and neural communication in the cerebral cortex. Electron microscopy (EM) studies in non-human model organisms have demonstrated that glutamate synaptic activity and functioning are directly reflected in quantifiable ultrastructural features. Thus, quantitative EM analysis of glutamate synapses in ex vivo preserved human brain tissue has the potential to provide novel insight into in vivo synaptic functioning. However, factors associated with the acquisition and preservation of human brain tissue have resulted in persistent concerns regarding the potential confounding effects of antemortem and postmortem biological processes on synaptic and sub-synaptic ultrastructural features. Thus, we sought to determine how well glutamate synaptic relationships and nanoarchitecture are preserved in postmortem human dorsolateral prefrontal cortex (DLPFC), a region that substantially differs in size and architecture from model systems. Focused ion beam-scanning electron microscopy (FIB-SEM), a powerful volume EM (VEM) approach, was employed to generate high-fidelity, fine-resolution, three-dimensional (3D) micrographic datasets appropriate for quantitative analyses. Using postmortem human DLPFC with a 6-hour postmortem interval, we optimized a tissue preservation and staining workflow that generated samples of excellent ultrastructural preservation and the high-contrast staining intensity required for FIB-SEM imaging. Quantitative analysis of sub-cellular, sub-synaptic and organelle components within glutamate axo-spinous synapses revealed that ultrastructural features of synaptic function and activity were well-preserved within and across individual synapses in postmortem human brain tissue. The synaptic, sub-synaptic and organelle measures were highly consistent with findings from experimental models that are free from antemortem or postmortem effects. Further, dense reconstruction of neuropil revealed a unique, ultrastructurally-complex, spiny dendritic shaft that exhibited features characteristic of neuronal processes with heightened synaptic communication, integration and plasticity. Altogether, our findings provide a critical proof-of-concept that ex vivo VEM analysis provides a valuable and informative means to infer in vivo functioning of human brain.

Aging disrupts the coordination between mRNA and protein expression in mouse and human midbrain.

Age-related dopamine (DA) neuron loss is a primary feature of Parkinson's disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Single nuclei transcriptomics in human and non-human primate striatum in opioid use disorder.

In brain, the striatum is a heterogenous region involved in reward and goal-directed behaviors. Striatal dysfunction is linked to psychiatric disorders, including opioid use disorder (OUD). Striatal subregions are divided based on neuroanatomy, each with unique roles in OUD. In OUD, the dorsal striatum is involved in altered reward processing, formation of habits, and development of negative affect during withdrawal. Using single nuclei RNA-sequencing, we identified both canonical (e.g., dopamine receptor subtype) and less abundant cell populations (e.g., interneurons) in human dorsal striatum. Pathways related to neurodegeneration, interferon response, and DNA damage were significantly enriched in striatal neurons of individuals with OUD. DNA damage markers were also elevated in striatal neurons of opioid-exposed rhesus macaques. Sex-specific molecular differences in glial cell subtypes associated with chronic stress were found in OUD, particularly female individuals. Together, we describe different cell types in human dorsal striatum and identify cell type-specific alterations in OUD.