Tight regulation of a nuclear HAPSTR1-HUWE1 pathway essential for mammalian life.

The recently discovered HAPSTR1 protein broadly oversees cellular stress responses. This function requires HUWE1, a ubiquitin ligase that paradoxically marks HAPSTR1 for degradation, but much about this pathway remains unclear. Here, leveraging multiplexed proteomics, we find that HAPSTR1 enables nuclear localization of HUWE1 with implications for nuclear protein quality control. We show that HAPSTR1 is tightly regulated and identify ubiquitin ligase TRIP12 and deubiquitinase USP7 as upstream regulators titrating HAPSTR1 stability. Finally, we generate conditional Hapstr1 knockout mice, finding that Hapstr1-null mice are perinatal lethal, adult mice depleted of Hapstr1 have reduced fitness, and primary cells explanted from Hapstr1-null animals falter in culture coincident with HUWE1 mislocalization and broadly remodeled signaling. Notably, although HAPSTR1 potently suppresses p53, we find that Hapstr1 is essential for life even in mice lacking p53. Altogether, we identify novel components and functional insights into the conserved HAPSTR1-HUWE1 pathway and demonstrate its requirement for mammalian life.

Neuron type-specific proteomics reveals distinct Shank3 proteoforms in iSPNs and dSPNs lead to striatal synaptopathy in Shank3B-/- mice.

Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies. To overcome the limitations associated with bulk measures of synaptic protein abundance, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Investigation of Shank3B-/- mice lacking exons 13-16 within the Shank3 gene, reveal distinct Shank3 protein isoform expression in iSPNs and dSPNs. In Shank3B-/- striatum, Shank3E and Shank3NT are expressed by dSPNs but are undetectable in iSPNs. Proteomic analysis indicates significant and selective alterations in the postsynaptic proteome of Shank3B-/- iSPNs. Correspondingly, the deletion of exons 13-16 diminishes dendritic spine density, reduces spine head diameter, and hampers corticostriatal synaptic transmission in iSPNs. Remarkably, reintroducing Shank3E in adult Shank3B-/- iSPNs significantly rectifies the observed dendritic spine morphological and corticostriatal synaptic transmission deficits. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.

Spatial neurolipidomics-MALDI mass spectrometry imaging of lipids in brain pathologies.

Given the complexity of nervous tissues, understanding neurochemical pathophysiology puts high demands on bioanalytical techniques with respect to specificity and sensitivity. Mass spectrometry imaging (MSI) has evolved to become an important, biochemical imaging technology for spatial biology in biological and translational research. The technique facilitates comprehensive, sensitive elucidation of the spatial distribution patterns of drugs, lipids, peptides, and small proteins in situ. Matrix-assisted laser desorption ionization (MALDI)-based MSI is the dominating modality due to its broad applicability and fair compromise of selectivity, sensitivity price, throughput, and ease of use. This is particularly relevant for the analysis of spatial lipid patterns, where no other comparable spatial profiling tools are available. Understanding spatial lipid biology in nervous tissue is therefore a key and emerging application area of MSI research. The aim of this review is to give a concise guide through the MSI workflow for lipid imaging in central nervous system (CNS) tissues and essential parameters to consider while developing and optimizing MSI assays. Further, this review provides a broad overview of key developments and applications of MALDI MSI-based spatial neurolipidomics to map lipid dynamics in neuronal structures, ultimately contributing to a better understanding of neurodegenerative disease pathology.

Local mitochondrial replication in the periphery of neurons requires the eEF1A1 protein and thetranslation of nuclear-encoded proteins.

In neurons, it is commonly assumed that mitochondrial replication only occurs in the cell body, after which the mitochondria must travel to the neuron's periphery. However, while mitochondrial DNA replication has been observed to occur away from the cell body, the specific mechanisms involved remain elusive. Using EdU-labelling in mouse primary neurons, we developed a tool to determine the mitochondrial replication rate. Taking of advantage of microfluidic devices, we confirmed that mitochondrial replication also occurs locally in the periphery of neurons. To achieve this, mitochondria require de novo nuclear-encoded, but not mitochondrial-encoded protein translation. Following a proteomic screen comparing synaptic with non-synaptic mitochondria, we identified two elongation factors - eEF1A1 and TUFM - that were upregulated in synaptic mitochondria. We found that mitochondrial replication is impaired upon the downregulation of eEF1A1, and this is particularly relevant in the periphery of neurons.

Notch receptor-ligand binding facilitates extracellular vesicle-mediated neuron-to-neuron communication.

Extracellular vesicles (EVs) facilitate intercellular communication by transferring cargo between cells in a variety of tissues. However, how EVs achieve cell-type-specific intercellular communication is still largely unknown. We found that Notch1 and Notch2 proteins are expressed on the surface of neuronal EVs that have been generated in response to neuronal excitatory synaptic activity. Notch ligands bind these EVs on the neuronal plasma membrane, trigger their internalization, activate the Notch signaling pathway, and drive the expression of Notch target genes. The generation of these neuronal EVs requires the endosomal sorting complex required for transport-associated protein Alix. Adult Alix conditional knockout mice have reduced hippocampal Notch signaling activation and glutamatergic synaptic protein expression. Thus, EVs facilitate neuron-to-neuron communication via the Notch receptor-ligand system in the brain.

Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Exceptional longevity of mammalian ovarian and oocyte macromolecules throughout the reproductive lifespan.

The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.

A Pathogenic Missense Mutation in Kainate Receptors Elevates Dendritic Excitability and Synaptic Integration through Dysregulation of SK Channels.

Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.

Amyloid fibril proteomics of AD brains reveals modifiers of aggregation and toxicity.

BACKGROUND: The accumulation of amyloid beta (Aß) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aß fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. METHODS: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aß immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aß42 flies, and Aß42 seeded cultured neurons. RESULTS: Amyloid fibrils are primarily composed by Aß42 and unexpectedly harbor Aß38 but generally lack Aß40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aß42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aß42 toxicity in vivo. CONCLUSIONS: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.

Parkinson's disease-linked parkin mutation disrupts recycling of synaptic vesicles in human dopaminergic neurons.

Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Early developmental deletion of forebrain Ank2 causes seizure-related phenotypes by reshaping the synaptic proteome.

Rare genetic variants in ANK2, which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2-/-:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2-/-:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2-/-:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2-/-:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments.

The cochlea is built to last a lifetime.

Orchestration of protein production and degradation and the regulation of protein lifetimes play a central role in many basic biological processes. Nearly all mammalian proteins are replenished by protein turnover in waves of synthesis and degradation. Protein lifetimes in vivo are typically measured in days, but a small number of extremely long-lived proteins (ELLPs) persist for months or even years. ELLPs are rare in all tissues but are enriched in tissues containing terminally differentiated post-mitotic cells and extracellular matrix. Consistently, emerging evidence suggests that the cochlea may be particularly enriched in ELLPs. Damage to ELLPs in specialized cell types, such as crystallin in the lens cells of the eye, causes organ failure such as cataracts. Similarly, damage to cochlear ELLPs is likely to occur with many insults, including acoustic overstimulation, drugs, anoxia, and antibiotics, and may play an underappreciated role in hearing loss. Furthermore, hampered protein degradation may contribute to acquired hearing loss. In this review, I highlight our knowledge of the lifetimes of cochlear proteins with an emphasis on ELLPs and the potential contribution that impaired cochlear protein degradation has on acquired hearing loss and the emerging relevance of ELLPs.

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. Significance: In this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.

Rescue of neuropsychiatric phenotypes in a mouse model of 16p11.2 duplication syndrome by genetic correction of an epilepsy network hub.

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers.

The TgF344-AD rat: behavioral and proteomic changes associated with aging and protein expression in a transgenic rat model of Alzheimer's disease.

Animal models of Alzheimer's Disease (AD) are attractive tools for preclinical, prodromal drug testing. The TgF344-AD (Tg) rat exhibits cognitive deficits and 5 major hallmarks of AD. Here we show that spatial water maze (WMZ) memory deficits and proteomic differences in dorsal CA1 were present in young Tg rats. Aged learning-unimpaired (AU) and aged learning-impaired (AI) proteome associated changes were identified and differed by sex. Levels of phosphorylated tau, reactive astrocytes and microglia were significantly increased in aged Tg rats and correlated with the WMZ learning index (LI); in contrast, no significant correlation was present between amyloid plaques or insoluble Aß levels and LI. Neuroinflammatory markers were also significantly correlated with LI and increased in female Tg rats. The anti-inflammatory marker, triggering receptor expressed on myeloid cells-2 (TREM2), was significantly reduced in aged impaired Tg rats and correlated with LI. Identifying and understanding mechanisms that allow for healthy aging by overcoming genetic drivers for AD, and/or promoting drivers for successful aging, are important for developing successful therapeutics against AD.

Determining and interpreting protein lifetimes in mammalian tissues.

The orchestration of protein production and degradation, and the regulation of protein lifetimes, play a central role in the majority of biological processes. Recent advances in proteomics have enabled the estimation of protein half-lives for thousands of proteins in vivo. What is the utility of these measurements, and how can they be leveraged to interpret the proteome changes occurring during development, aging, and disease? This opinion article summarizes leading technical approaches and highlights their strengths and weaknesses. We also disambiguate frequently used terminology, illustrate recent mechanistic insights, and provide guidance for interpreting and validating protein turnover measurements. Overall, protein lifetimes, coupled to estimates of protein levels, are essential for obtaining a deep understanding of mammalian biology and the basic processes defining life itself.

Cochlear ribbon synapse maturation requires Nlgn1 and Nlgn3.

Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.

Electron Tomographic Methods for Studying Organelles of the Murine Chemical Synapse.

Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.