Jerrel L. Yakel

Viral infections are common across the human lifespan, yet their molecular effects on human neurons remain poorly characterized. We present a mass spectrometry (MS)-based proteomic dataset profiling human induced pluripotent stem cell (iPSC)-derived neurons following exposure to five neurotropic viruses: Herpes simplex virus 1 (HSV-1), Human coronavirus 229E (HCoV-229E), Epstein-Barr virus (EBV), Varicella-Zoster virus (VZV), and Influenza A virus (H1N1). Neurons differentiated from the KOLF2.1 J iPSC line were infected at multiple viral doses and sampled at three post-infection time points. Protein abundance was quantified using data-independent acquisition mass spectrometry and a customized human-viral proteome library, enabling simultaneous detection of host and viral proteins. The dataset includes approximately 7,500 human proteins and virus-specific peptides, providing a systematically controlled resource to compare virus- and time-dependent proteomic responses. All raw MS files and processed data are publicly deposited in the PRIDE repository, and an interactive web application supports data exploration and reuse. This dataset is designed to support cross-virus comparisons, hypothesis generation, and integration with transcriptomics and population-scale datasets.

In the central nervous system, acetylcholine (ACh) neurons coordinate neural network activity required for higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbances in cholinergic signaling have been described in many diseases of the developing and mature brain. Interestingly, ACh neurons can co-transmit GABA to support essential roles in brain function. However, the contributions of ACh/GABA co-transmission to brain function remain unclear. This underscores the need to better understand the heterogeneity of ACh neurons, particularly the sub-population of ACh neurons co-expressing GABAergic markers. We used various combinations of transgenic mouse lines to systematically label ACh neuron populations positive for different GABAergic markers in the brain. We developed a workflow combining tissue clearing, light-sheet fluorescence microscopy, and machine learning to image entire mouse brain hemispheres followed by quantification of ACh neurons throughout the brain. With this approach, we assessed the role of GABA co-transmission in ACh neuron function and quantified ACh and ACh/GABA neuron sub-populations in the brain. Our results suggest that GABA co-transmission from ACh neurons is not required to maintain the regular ACh neuron count in the brain. Furthermore, we report that a large subset of ACh neurons can potentially synthesize GABA by co-expressing the marker Gad2. However, most of these ACh neurons do not express vGAT, which would enable these neurons to release GABA. Taken together, we conclude that GABA co-transmission likely occurs only from a small population of ACh neurons restricted to few brain nuclei.

Endemic viral infections with low pathogenicity are often overlooked due to their mild symptoms, yet they can exert long-term effects on cellular function and contribute to disease pathogenesis. While viral infections have been implicated in neurodegenerative disorders, their impact on the neuronal proteome remains poorly understood. Here, we differentiated human induced pluripotent stem cells (KOLF2.1J) into mature neurons to investigate virus-induced proteomic changes following infection with five neurotropic endemic human viruses: Herpes simplex virus 1 (HSV-1), Human coronavirus 229E (HCoV-229E), Epstein-Barr virus (EBV), Varicella-Zoster virus (VZV), and Influenza A virus (H1N1). Given that these viruses can infect adults and have the potential to cross the placental barrier, their molecular impact on neurons may be relevant across the lifespan. Using mass spectrometry-based proteomics with a customized library for simultaneous detection of human and viral proteins, we confirmed successful infections and identified virus-specific proteomic signatures. Notably, virus-induced protein expression changes converged on key neuronal pathways, including those associated with neurodegeneration. Gene co-expression network analysis identified protein modules correlated with viral proteins. Pathway enrichment analysis of these modules revealed associations with the nervous system, including pathways linked to Alzheimer’s and Parkinson’s disease. Remarkably, several viral-induced proteomic alterations overlapped with changes observed in postmortem Alzheimer’s patient brains, suggesting a mechanistic connection between viral exposure and neurodegenerative disease progression. These findings provide molecular insights into how common viral infections perturb neuronal homeostasis and may contribute to neurodegenerative pathology, highlighting the need to consider endemic viruses as potential environmental risk factors in neurological disorders.

Hippocamposeptal (HS) neurons send GABAergic projections from the hippocampus to the medial septum/diagonal band of Broca (MS/DBB) as part of a reciprocal loop that is critical for memory. HS neurons are proposed to be particularly sensitive to the deleterious effects of pathological exposure to amyloid-β (Aβ), as would occur during Alzheimer’s disease (AD). However, it is not known how HS GABA release in the MS/DBB is altered during the progression of AD. To target HS neurons in a mouse model of AD, we crossed SST-Cre mice to 5XFAD mice and performed stereotaxic injections of Cre-dependent AAV containing mCherry/channelrhodopsin-2 (ChR2) into the hippocampus of offspring at 4, 6, 9, and 12 months. We used optogenetics to selectively stimulate HS terminals while performing whole-cell patch-clamp recordings from MS/DBB neurons in slices. There was a transient reduction in HS-inhibitory postsynaptic current (IPSC) amplitude in female 5XFAD mice at 6 months, but no difference in males at any age, and no difference in paired-pulse ratio in either sex at any age. When bath applying the GABA_BR agonist, baclofen, we found a larger decrease in HS-IPSC amplitude in 5XFAD females at 9 months and 5XFAD males at 12 months. In 12-month-old 5XFAD females, response to baclofen was significantly reduced. These data suggest that there is a transient increase in responsiveness to GABA_BR activation in 5XFAD mice that occurs earlier in females than in males. These sex-specific changes to HS function are likely to impact the relay of information between the hippocampus and MS/DBB.

Acetylcholine (ACh) neurons in the central nervous system are required for the coordination of neural network activity during higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbed cholinergic signaling has been described in many neurodevelopmental and neurodegenerative disorders. Furthermore, cotransmission of other signaling molecules, such as glutamate and GABA, with ACh has been associated with essential roles in brain function or disease. However, it is unknown when ACh neurons become cholinergic during development. Thus, understanding the timeline of how the cholinergic system develops and becomes active in the healthy brain is a crucial part of understanding brain development. To study this, we used transgenic mice to selectively label ACh neurons with tdTomato. We imaged serial sectioned brains and generated whole-brain reconstructions at different time points during pre- and postnatal development. We found three crucial time windows—two in the prenatal and one in the postnatal brain—during which most ACh neuron populations become cholinergic in the brain. We also found that cholinergic gene expression is initiated in cortical ACh interneurons, while the cerebral cortex is innervated by cholinergic projection neurons from the basal forebrain. Taken together, we show that ACh neuron populations are present and become cholinergic before postnatal day 12, which is the onset of major sensory processes, such as hearing and vision. We conclude that the birth of ACh neurons and initiation of cholinergic gene expression are temporally separated during development but highly coordinated by brain anatomical structure.

Cholinergic regulation of hippocampal theta oscillations has long been proposed to be a potential mechanism underlying hippocampus-dependent memory encoding processes. However, cholinergic transmission has been traditionally associated with type II theta under urethane anesthesia. The mechanisms and behavioral significance of cholinergic regulation of type I theta in freely exploring animals is much less clear. In this study, we examined the potential behavioral significance of cholinergic regulation of theta oscillations in the object location task in male mice that involves training and testing trials and provides an ideal behavioral task to study the underlying memory encoding and retrieval processes, respectively. Cholinergic regulation of hippocampal theta oscillations and the behavioral outcomes was examined by either intrahippocampal infusion of cholinergic receptor antagonists or knocking out cholinergic receptors in excitatory neurons or interneurons. We found that both muscarinic acetylcholine receptors (mAChRs) and α7 nicotinic AChRs (α7 nAChRs) regulated memory encoding by engaging excitatory neurons and interneurons, respectively. There is a transient upregulated theta oscillation at the beginning of individual object exploration events that only occurred in the training trials, but not in the testing trials. This transient upregulated theta is also the only theta component that significantly differed between training and testing trials and was sensitive to mAChR and α7 nAChR antagonists. Thus, our study has revealed a transient cholinergic-sensitive theta component that is specifically associated with memory encoding, but not memory retrieval, in the object location task, providing direct experimental evidence supporting a role for cholinergic-regulated theta oscillations in hippocampus-dependent memory encoding processes.Significance statementIn this study, we made several important findings regarding cholinergic mechanisms regulating hippocampal memory formation in the object location task. Firstly, both hippocampal mAChRs and the α7 nAChRs regulated the memory encoding process in the object location task by engaging glutamatergic neurons and interneurons, respectively. Secondly, upregulated theta oscillations were observed in the pre-exploration period in training trials, but not in the testing trials, suggesting a role in novelty encoding/learning. Thirdly, the upregulated theta was sensitive to mAChR and α7 nAChR antagonists and receptor knockouts in neuronal subpopulations. Taken together, these observations provide direct experimental evidence supporting a role for cholinergic-regulated theta oscillations in hippocampal memory encoding process.