Plasma and CSF biomarkers of aging and cognitive decline in Caribbean vervets.

INTRODUCTION: Vervets are non-human primates that share high genetic homology with humans and develop amyloid beta (Aß) pathology with aging. We expand current knowledge by examining Aß pathology, aging, cognition, and biomarker proteomics. METHODS: Amyloid immunoreactivity in the frontal cortex and temporal cortex/hippocampal regions from archived vervet brain samples ranging from young adulthood to old age was quantified. We also obtained cognitive scores, plasma samples, and cerebrospinal fluid (CSF) samples in additional animals. Plasma and CSF proteins were quantified with platforms utilizing human antibodies. RESULTS: We found age-related increases in Aß deposition in both brain regions. Bioinformatic analyses assessed associations between biomarkers and age, sex, cognition, and CSF Aß levels, revealing changes in proteins related to immune-related inflammation, metabolism, and cellular processes. DISCUSSION: Vervets are an effective model of aging and early-stage Alzheimer's disease, and we provide translational biomarker data that both align with previous results in humans and provide a basis for future investigations. HIGHLIGHTS: We found changes in immune and metabolic plasma biomarkers associated with age and cognition. Cerebrospinal fluid (CSF) biomarkers revealed changes in cell signaling indicative of adaptative processes. TNFRSF19 (TROY) and Artemin co-localize with Alzheimer's disease pathology. Vervets are a relevant model for translational studies of early-stage Alzheimer's disease.

Peripheral B cells repress B-cell regeneration in aging through a TNF-α/IGFBP-1/IGF-1 immune-endocrine axis.

Loss of B lymphocyte regeneration in the bone marrow (BM) is an immunologic hallmark of advanced age, which impairs the replenishment of peripheral B-cell subsets and results in impaired humoral responses, thereby contributing to immune system dysfunction associated with aging. A better understanding of the mechanism behind this loss may suggest ways to restore immune competence and promote healthy aging. In this study, we uncover an immune-endocrine regulatory circuit that mediates cross-talk between peripheral B cells and progenitors in the BM, to balance B-cell lymphopoiesis in both human and mouse aging. We found that tumor necrosis factor α (TNF-α), which is increasingly produced by peripheral B cells during aging, stimulates the production of insulin-like growth factor-binding protein 1 (IGFBP-1), which binds and sequesters insulin-like growth factor 1 (IGF-1) in the circulation, thereby restraining its activity in promoting B-cell lymphopoiesis in the BM. Upon B-cell depletion in aging humans and mice, circulatory TNF-α decreases, resulting in increased IGF-1 and reactivation of B-cell lymphopoiesis. Perturbation of this circuit by administration of IGF-1 to old mice or anti-TNF-α antibodies to human patients restored B-cell lymphopoiesis in the BM. Thus, we suggest that in both human and mouse aging, peripheral B cells use the TNF-α/IGFBP-1/IGF-1 axis to repress B-cell lymphopoiesis. This trial was registered at www.clinicaltrials.govas#NCT00863187.

Dysregulation of brain and choroid plexus cell types in severe COVID-19.

Although SARS-CoV-2 primarily targets the respiratory system, patients with and survivors of COVID-19 can suffer neurological symptoms1-3. However, an unbiased understanding of the cellular and molecular processes that are affected in the brains of patients with COVID-19 is missing. Here we profile 65,309 single-nucleus transcriptomes from 30 frontal cortex and choroid plexus samples across 14 control individuals (including 1 patient with terminal influenza) and 8 patients with COVID-19. Although our systematic analysis yields no molecular traces of SARS-CoV-2 in the brain, we observe broad cellular perturbations indicating that barrier cells of the choroid plexus sense and relay peripheral inflammation into the brain and show that peripheral T cells infiltrate the parenchyma. We discover microglia and astrocyte subpopulations associated with COVID-19 that share features with pathological cell states that have previously been reported in human neurodegenerative disease4-6. Synaptic signalling of upper-layer excitatory neurons-which are evolutionarily expanded in humans7 and linked to cognitive function8-is preferentially affected in COVID-19. Across cell types, perturbations associated with COVID-19 overlap with those found in chronic brain disorders and reside in genetic variants associated with cognition, schizophrenia and depression. Our findings and public dataset provide a molecular framework to understand current observations of COVID-19-related neurological disease, and any such disease that may emerge at a later date.

A neuronal blood marker is associated with mortality in old age.

Neurofilament light chain (NfL) has emerged as a promising blood biomarker for the progression of various neurological diseases. NfL is a structural protein of nerve cells, and elevated NfL levels in blood are thought to mirror damage to the nervous system. We find that plasma NfL levels increase in humans with age (n = 122; 21-107 years of age) and correlate with changes in other plasma proteins linked to neural pathways. In centenarians (n = 135), plasma NfL levels are associated with mortality equally or better than previously described multi-item scales of cognitive or physical functioning, and this observation was replicated in an independent cohort of nonagenarians (n = 180). Plasma NfL levels also increase in aging mice (n = 114; 2-30 months of age), and dietary restriction, a paradigm that extends lifespan in mice, attenuates the age-related increase in plasma NfL levels. These observations suggest a contribution of nervous system functional deterioration to late-life mortality.

Ageing hallmarks exhibit organ-specific temporal signatures.

Ageing is the single greatest cause of disease and death worldwide, and understanding the associated processes could vastly improve quality of life. Although major categories of ageing damage have been identified-such as altered intercellular communication, loss of proteostasis and eroded mitochondrial function1-these deleterious processes interact with extraordinary complexity within and between organs, and a comprehensive, whole-organism analysis of ageing dynamics has been lacking. Here we performed bulk RNA sequencing of 17 organs and plasma proteomics at 10 ages across the lifespan of Mus musculus, and integrated these findings with data from the accompanying Tabula Muris Senis2-or 'Mouse Ageing Cell Atlas'-which follows on from the original Tabula Muris3. We reveal linear and nonlinear shifts in gene expression during ageing, with the associated genes clustered in consistent trajectory groups with coherent biological functions-including extracellular matrix regulation, unfolded protein binding, mitochondrial function, and inflammatory and immune response. Notably, these gene sets show similar expression across tissues, differing only in the amplitude and the age of onset of expression. Widespread activation of immune cells is especially pronounced, and is first detectable in white adipose depots during middle age. Single-cell RNA sequencing confirms the accumulation of T cells and B cells in adipose tissue-including plasma cells that express immunoglobulin J-which also accrue concurrently across diverse organs. Finally, we show how gene expression shifts in distinct tissues are highly correlated with corresponding protein levels in plasma, thus potentially contributing to the ageing of the systemic circulation. Together, these data demonstrate a similar yet asynchronous inter- and intra-organ progression of ageing, providing a foundation from which to track systemic sources of declining health at old age.

Physiological blood-brain transport is impaired with age by a shift in transcytosis.

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.

Undulating changes in human plasma proteome profiles across the lifespan.

Aging is a predominant risk factor for several chronic diseases that limit healthspan1. Mechanisms of aging are thus increasingly recognized as potential therapeutic targets. Blood from young mice reverses aspects of aging and disease across multiple tissues2-10, which supports a hypothesis that age-related molecular changes in blood could provide new insights into age-related disease biology. We measured 2,925 plasma proteins from 4,263 young adults to nonagenarians (18-95 years old) and developed a new bioinformatics approach that uncovered marked non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh and eighth decades of life reflected distinct biological pathways and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits. This new approach to the study of aging led to the identification of unexpected signatures and pathways that might offer potential targets for age-related diseases.

Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics.

Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice.

Ageing drives changes in neuronal and cognitive function, the decline of which is a major feature of many neurological disorders. The hippocampus, a brain region subserving roles of spatial and episodic memory and learning, is sensitive to the detrimental effects of ageing at morphological and molecular levels. With advancing age, synapses in various hippocampal subfields exhibit impaired long-term potentiation, an electrophysiological correlate of learning and memory. At the molecular level, immediate early genes are among the synaptic plasticity genes that are both induced by long-term potentiation and downregulated in the aged brain. In addition to revitalizing other aged tissues, exposure to factors in young blood counteracts age-related changes in these central nervous system parameters, although the identities of specific cognition-promoting factors or whether such activity exists in human plasma remains unknown. We hypothesized that plasma of an early developmental stage, namely umbilical cord plasma, provides a reservoir of such plasticity-promoting proteins. Here we show that human cord plasma treatment revitalizes the hippocampus and improves cognitive function in aged mice. Tissue inhibitor of metalloproteinases 2 (TIMP2), a blood-borne factor enriched in human cord plasma, young mouse plasma, and young mouse hippocampi, appears in the brain after systemic administration and increases synaptic plasticity and hippocampal-dependent cognition in aged mice. Depletion experiments in aged mice revealed TIMP2 to be necessary for the cognitive benefits conferred by cord plasma. We find that systemic pools of TIMP2 are necessary for spatial memory in young mice, while treatment of brain slices with TIMP2 antibody prevents long-term potentiation, arguing for previously unknown roles for TIMP2 in normal hippocampal function. Our findings reveal that human cord plasma contains plasticity-enhancing proteins of high translational value for targeting ageing- or disease-associated hippocampal dysfunction.

Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice.

As human lifespan increases, a greater fraction of the population is suffering from age-related cognitive impairments, making it important to elucidate a means to combat the effects of aging. Here we report that exposure of an aged animal to young blood can counteract and reverse pre-existing effects of brain aging at the molecular, structural, functional and cognitive level. Genome-wide microarray analysis of heterochronic parabionts--in which circulatory systems of young and aged animals are connected--identified synaptic plasticity-related transcriptional changes in the hippocampus of aged mice. Dendritic spine density of mature neurons increased and synaptic plasticity improved in the hippocampus of aged heterochronic parabionts. At the cognitive level, systemic administration of young blood plasma into aged mice improved age-related cognitive impairments in both contextual fear conditioning and spatial learning and memory. Structural and cognitive enhancements elicited by exposure to young blood are mediated, in part, by activation of the cyclic AMP response element binding protein (Creb) in the aged hippocampus. Our data indicate that exposure of aged mice to young blood late in life is capable of rejuvenating synaptic plasticity and improving cognitive function.

APOE ε4 worsens hippocampal CA1 apical neuropil atrophy and episodic memory.

OBJECTIVES: Using high-resolution structural MRI, we endeavored to study the relationships among APOE ε4, hippocampal subfield and stratal anatomy, and episodic memory. METHODS: Using a cross-sectional design, we studied 11 patients with Alzheimer disease dementia, 14 patients with amnestic mild cognitive impairment, and 14 age-matched healthy controls with no group differences in APOE ε4 carrier status. Each subject underwent ultra-high-field 7.0-tesla MRI targeted to the hippocampus and neuropsychological assessment. RESULTS: We found a selective, dose-dependent association of APOE ε4 with greater thinning of the CA1 apical neuropil, or stratum radiatum/stratum lacunosum-moleculare (CA1-SRLM), a hippocampal subregion known to exhibit early vulnerability to neurofibrillary pathology in Alzheimer disease. The relationship between the ε4 allele and CA1-SRLM thinning persisted after controlling for dementia severity, and the size of other hippocampal subfields and the entorhinal cortex did not differ by APOE ε4 carrier status. Carriers also exhibited worse episodic memory function but similar performance in other cognitive domains compared with noncarriers. In a statistical mediation analysis, we found support for the hypothesis that CA1-SRLM thinning may link the APOE ε4 allele to its phenotypic effects on memory. CONCLUSIONS: The APOE ε4 allele segregated dose-dependently and selectively with CA1-SRLM thinning and worse episodic memory performance in a pool of older subjects across a cognitive spectrum. These findings highlight a possible role for this gene in influencing a critical hippocampal subregion and an associated symptomatic manifestation.

The sox gene Dichaete is expressed in local interneurons and functions in development of the Drosophila adult olfactory circuit.

In insects, the primary sites of integration for olfactory sensory input are the glomeruli in the antennal lobes. Here, axons of olfactory receptor neurons synapse with dendrites of the projection neurons that relay olfactory input to higher brain centers, such as the mushroom bodies and lateral horn. Interactions between olfactory receptor neurons and projection neurons are modulated by excitatory and inhibitory input from a group of local interneurons. While significant insight has been gleaned into the differentiation of olfactory receptor and projection neurons, much less is known about the development and function of the local interneurons. We have found that Dichaete, a conserved Sox HMG box gene, is strongly expressed in a cluster of LAAL cells located adjacent to each antennal lobe in the adult brain. Within these clusters, Dichaete protein expression is detected in both cholinergic and GABAergic local interneurons. In contrast, Dichaete expression is not detected in mature or developing projection neurons, or developing olfactory receptor neurons. Analysis of novel viable Dichaete mutant alleles revealed misrouting of specific projection neuron dendrites and axons, and alterations in glomeruli organization. These results suggest noncell autonomous functions of Dichaete in projection neuron differentiation as well as a potential role for Dichaete-expressing local interneurons in development of the adult olfactory circuitry.

The SUMO protease Verloren regulates dendrite and axon targeting in olfactory projection neurons.

Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and deconjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. Here, we show a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. We uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO deconjugation rather than in maturation. Our study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival.

MicroRNA processing pathway regulates olfactory neuron morphogenesis.

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs [1, 2]. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes [3, 4]. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition (Figure 1A). Here, we identify two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections [5, 6]. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers [7-9]. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendrite mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. Our findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system.

piggyBac-based mosaic screen identifies a postmitotic function for cohesin in regulating developmental axon pruning.

Developmental axon pruning is widely used to refine neural circuits. We performed a mosaic screen to identify mutations affecting axon pruning of Drosophila mushroom body gamma neurons. We constructed a modified piggyBac vector with improved mutagenicity and generated insertions in >2000 genes. We identified two cohesin subunits (SMC1 and SA) as being essential for axon pruning. The cohesin complex maintains sister-chromatid cohesion during cell division in eukaryotes. However, we show that the pruning phenotype in SMC1(-/-) clones is rescued by expressing SMC1 in neurons, revealing a postmitotic function. SMC1(-/-) clones exhibit reduced levels of the ecdysone receptor EcR-B1, a key regulator of axon pruning. The pruning phenotype is significantly suppressed by overexpressing EcR-B1 and is enhanced by a reduced dose of EcR, supporting a causal relationship. We also demonstrate a postmitotic role for SMC1 in dendrite targeting of olfactory projection neurons. We suggest that cohesin regulates diverse aspects of neuronal morphogenesis.

Temporal target restriction of olfactory receptor neurons by Semaphorin-1a/PlexinA-mediated axon-axon interactions.

Axon-axon interactions have been implicated in neural circuit assembly, but the underlying mechanisms are poorly understood. Here, we show that in the Drosophila antennal lobe, early-arriving axons of olfactory receptor neurons (ORNs) from the antenna are required for the proper targeting of late-arriving ORN axons from the maxillary palp (MP). Semaphorin-1a is required for targeting of all MP but only half of the antennal ORN classes examined. Sema-1a acts nonautonomously to control ORN axon-axon interactions, in contrast to its cell-autonomous function in olfactory projection neurons. Phenotypic and genetic interaction analyses implicate PlexinA as the Sema-1a receptor in ORN targeting. Sema-1a on antennal ORN axons is required for correct targeting of MP axons within the antennal lobe, while interactions amongst MP axons facilitate their entry into the antennal lobe. We propose that Sema-1a/PlexinA-mediated repulsion provides a mechanism by which early-arriving ORN axons constrain the target choices of late-arriving axons.

Mitotic activation of the kinase Aurora-A requires its binding partner Bora.

The protein kinase Aurora-A is required for centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis. Here, we describe the identification of Bora, a conserved protein that is required for the activation of Aurora-A at the onset of mitosis. In the Drosophila peripheral nervous system, bora mutants have defects during asymmetric cell division identical to those observed in aurora-A. Furthermore, overexpression of bora can rescue defects caused by mutations in aurora-A. Bora is conserved in vertebrates, and both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro. In interphase cells, Bora is a nuclear protein, but upon entry into mitosis, Bora is excluded from the nucleus and translocates into the cytoplasm in a Cdc2-dependent manner. We propose a model in which activation of Cdc2 initiates the release of Bora into the cytoplasm where it can bind and activate Aurora-A.

Wiring stability of the adult Drosophila olfactory circuit after lesion.

Neuronal wiring plasticity in response to experience or injury has been reported in many parts of the adult nervous system. For instance, visual or somatosensory cortical maps can reorganize significantly in response to peripheral lesions, yet a certain degree of stability is essential for neuronal circuits to perform their dedicated functions. Previous studies on lesion-induced neuronal reorganization have primarily focused on systems that use continuous neural maps. Here, we assess wiring plasticity in a discrete neural map represented by the adult Drosophila olfactory circuit. Using conditional expression of toxins, we genetically ablated specific classes of neurons and examined the consequences on their synaptic partners or neighboring classes in the adult antennal lobe. We find no alteration of connection specificity between olfactory receptor neurons (ORNs) and their postsynaptic targets, the projection neurons (PNs). Ablating an ORN class maintains PN dendrites within their glomerular borders, and ORN axons normally innervating an adjacent target do not expand. Likewise, ablating PN classes does not alter their partner ORN axon connectivity. Interestingly, an increase in the contralateral ORN axon terminal density occurs in response to the removal of competing ipsilateral ORNs. Therefore, plasticity in this circuit can occur but is confined within a glomerulus, thereby retaining the wiring specificity of ORNs and PNs. We conclude that, although adult olfactory neurons can undergo plastic changes in response to the loss of competition, the olfactory circuit overall is extremely stable in preserving segregated information channels in this discrete map.

Dendritic patterning by Dscam and synaptic partner matching in the Drosophila antennal lobe.

In the olfactory system of Drosophila melanogaster, axons of olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons typically target 1 of approximately 50 glomeruli. Dscam, an immunoglobulin superfamily protein, acts in ORNs to regulate axon targeting. Here we show that Dscam acts in projection neurons and local interneurons to control the elaboration of dendritic fields. The removal of Dscam selectively from projection neurons or local interneurons led to clumped dendrites and marked reduction in their dendritic field size. Overexpression of Dscam in projection neurons caused dendrites to be more diffuse during development and shifted their relative position in adulthood. Notably, the positional shift of projection neuron dendrites caused a corresponding shift of its partner ORN axons, thus maintaining the connection specificity. This observation provides evidence for a pre- and postsynaptic matching mechanism independent of precise glomerular positioning.