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Acetylcholinesterase (AChE) is a highly conserved enzyme responsible for the regulation of acetylcholine signaling within the brain and periphery. AChE has also been shown to participate in non-enzymatic activity and contribute to cellular development and aging. In particular, enzymatic cleavage of the synaptic AChE isoform, AChE-T, is shown to generate a bioactive T30 peptide that binds to the ⍺7 nicotinic acetylcholine receptor (nAChR) at synapses. Here, we explore intracellular mechanisms of T30 signaling within the human cholinergic neural cell line SH-SY5Y using high performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS). Proteomic analysis of cells exposed to (100 nM) T30 for 3-days reveals significant changes within proteins important for cell growth. Specifically, bioinformatic analysis identifies proteins that converge onto the mammalian target of rapamycin (mTOR) pathway signaling. Functional experiments confirm that T30 regulates neural cell growth via mTOR signaling and ⍺7 nAChR activation. T30 was found promote mTORC1 pro-growth signaling through an increase in phosphorylated elF4E and S6K1, and a decrease in the autophagy LC3B-II protein. These findings are corroborated in hippocampal neurons and show that T30 promotes dendritic arborization. Taken together, our findings define mTOR as a novel pathway activated by T30 interaction with the nAChR and suggest a role for this process in human disease.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using singlecell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson’s disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2 G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague–Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by amyloid beta (Ab) and neurofibrillary tangles (NFTs) containing the microtubule associated protein tau. Tau pathology is highly correlated to neuronal loss, unlike plaque load. Tau likely acts as a downstream effector of Ab, leading to neurotoxicity. Indeed, the loss of tau can rescue neurotoxicity. Furthermore, aggregated tau propagates in a prion-like manner, initiating a toxic cascade. It has been shown that its release is exacerbated via excitation. Thus, tau-directed approaches for novel treatments of AD are essential. The hyperphosphorylation of tau is seen in all tauopathies, not exclusively AD, suggesting an essential role in pathogenesis. Physiologically, tau is unfolded, suggesting that aggregated, phosphorylated tau is pathological. To understand neurodegenerative pathways, it is imperative to study the pattern of tau phosphorylation. However, there are over 80 potential phosphorylation sites on tau resulting in an ambiguous definition of “phosphorylated tau”. We are particularly interested in tau’s role in hyperexcitation because aberrant overactivity is a characteristic of AD as well as other tauopathies and may be via catalyst for further pathogenesis. Here, we used unbiased mass spectrometry(MS) to perform comprehensive mapping of tau phosphorylation patterns during hyperexcitation. We have identified a unique signature of tau phosphorylation during hyperexcitation S409, S412, S413, T414, S416, S422,and T427. Subsequently, we mutated each of these to phosphor-mimetic or phosphor-null sites to identify any changes associated with tau localization, neuronal morphology, and spine morphology. We found that individual sites display different phenotypes of tau, effecting tau intensity at particular subcellular locations and neuronal branching. Lastly, we identified the phosphorylation patterns over time in various mouse models. We found overtime tau phosphorylation patterns differ between sex and age. These results will begin to decipher the phosphorylation code that governs tau function in physiology and pathology. Defining the phosphorylation profile of tau is highly significant for understanding basic mechanisms involved in hyperexcitation and disease progression in neurodegeneration. This work will help guide new therapeutic approaches for targeting pathogenic tau in tauopathies such as AD.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using singlecell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson’s disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2 G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using singlecell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2.sup.G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using singlecell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2 knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Addresses reviewer comments. text updated, small changes made to main and supplementary figures* https://dopabase.org

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2 knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

The end-stage pathology of Parkinson's disease (PD) involves the loss of dopamine-producing neurons in the substantia nigra pars compacta (SNc). However, synaptic deregulation of these neurons begins much earlier. Understanding the mechanisms behind synaptic deficits is crucial for early therapeutic intervention, yet these remain largely unknown. In the SNc, different dopamine neuron subtypes show varying susceptibility patterns to PD, complicating our understanding. This study uses intersectional genetic mouse models to uncover synaptic perturbations in vulnerable dopamine neurons, focusing on the LRRK2 kinase, a protein closely linked to PD. Through a combination of immunofluorescence and advanced proximity labeling methods, we found higher LRRK2 expression in the most vulnerable dopamine neuron subclusters. High-resolution imaging revealed that pathogenic LRRK2 disrupts release sites in vulnerable dopamine axons, leading to decreased in vivo evoked striatal dopamine release in mice with LRRK2 mutations. Proteomic and biochemical analyses indicate that mutant LRRK2 increases the phosphorylation of RAB3 proteins, reducing their interactions with RIM1/2 effector proteins and impacting their synaptic functions. Overall, this research highlights the cell-autonomous dysfunctions caused by mutant LRRK2 in the neurons that are primarily affected by the disease. It also provides a framework for therapeutic strategies for early nigrostriatal synaptic deficits in PD.