The evolution of lineage-specific regulatory activities in the human embryonic limb.

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.

Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here, we show that FMRP regulates translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca's area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases, but not FMRP-deficient mice. Thus, alterations in FMRP posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

Origin and development of the claustrum in rhesus macaque.

Understanding the claustrum's functions has recently progressed thanks to new anatomical and behavioral studies in rodents, which suggest that it plays an important role in attention, salience detection, slow-wave generation, and neocortical network synchronization. Nevertheless, knowledge about the origin and development of the claustrum, especially in primates, is still limited. Here, we show that neurons of rhesus macaque claustrum primordium are generated between embryonic day E48 and E55 and express some neocortical molecular markers, such as NR4A2, SATB2, and SOX5. However, in the early stages, it lacks TBR1 expression, which separates it from other surrounding telencephalic structures. We also found that two waves of neurogenesis (E48 and E55) in the claustrum, corresponding to the birthdates of layers 6 and 5 of the insular cortex, establish a "core" and "shell" cytoarchitecture, which is potentially a basis for differential circuit formation and could influence information processing underlying higher cognitive functions of the claustrum. In addition, parvalbumin-positive interneurons are the dominant interneuron type in the claustrum in fetal macaque, and their maturation is independent of that in the overlaying neocortex. Finally, our study reveals that the claustrum is likely not a continuance of subplate neurons of the insular cortex, but an independent pallial region, suggesting its potentially unique role in cognitive control.

Role of intracortical neuropil growth in the gyrification of the primate cerebral cortex.

The convolutions of the mammalian cerebral cortex allow the enlargement of its surface and addition of novel functional areas during evolution while minimizing expansion of the cranium. Cognitive neurodevelopmental disorders in humans, including microcephaly and lissencephaly, are often associated with impaired gyrification. In the classical model of gyrification, surface area is initially set by the number of radial units, and the forces driving cortical folding include neuronal growth, formation of neuropil, glial cell intercalation, and the patterned growth of subcortical white matter. An alternative model proposes that specified neurogenic hotspots in the outer subventricular zone (oSVZ) produce larger numbers of neurons that generate convexities in the cortex. This directly contradicts reports showing that cortical neurogenesis and settling of neurons into the cortical plate in primates, including humans, are completed well prior to the formation of secondary and tertiary gyri and indeed most primary gyri. In addition, during the main period of gyrification, the oSVZ produces mainly astrocytes and oligodendrocytes. Here we describe how rapid growth of intracortical neuropil, addition of glial cells, and enlargement of subcortical white matter in primates are the primary forces responsible for the post-neurogenic expansion of the cortical surface and formation of gyri during fetal development. Using immunohistochemistry for markers of proliferation and glial and neuronal progenitors combined with transcriptomic analysis, we show that neurogenesis in the ventricular zone and oSVZ is phased out and transitions to gliogenesis prior to gyral development. In summary, our data support the classical model of gyrification and provide insight into the pathogenesis of congenital cortical malformations.

Alternative splicing events as peripheral biomarkers for motor learning deficit caused by adverse prenatal environments.

Severity of neurobehavioral deficits in children born from adverse pregnancies, such as maternal alcohol consumption and diabetes, does not always correlate with the adversity's duration and intensity. Therefore, biological signatures for accurate prediction of the severity of neurobehavioral deficits, and robust tools for reliable identification of such biomarkers, have an urgent clinical need. Here, we demonstrate that significant changes in the alternative splicing (AS) pattern of offspring lymphocyte RNA can function as accurate peripheral biomarkers for motor learning deficits in mouse models of prenatal alcohol exposure (PAE) and offspring of mother with diabetes (OMD). An aptly trained deep-learning model identified 29 AS events common to PAE and OMD as superior predictors of motor learning deficits than AS events specific to PAE or OMD. Shapley-value analysis, a game-theory algorithm, deciphered the trained deep-learning model's learnt associations between its input, AS events, and output, motor learning performance. Shapley values of the deep-learning model's input identified the relative contribution of the 29 common AS events to the motor learning deficit. Gene ontology and predictive structure-function analyses, using Alphafold2 algorithm, supported existing evidence on the critical roles of these molecules in early brain development and function. The direction of most AS events was opposite in PAE and OMD, potentially from differential expression of RNA binding proteins in PAE and OMD. Altogether, this study posits that AS of lymphocyte RNA is a rich resource, and deep-learning is an effective tool, for discovery of peripheral biomarkers of neurobehavioral deficits in children of diverse adverse pregnancies.

The analysis of H.M.'s brain: A brief review of status and plans for future studies and tissue archive.

The famous amnesic patient Henry Molaison (H.M.) died on December 2, 2008. After extensive in situ magnetic resonance imaging in Boston, his brain was removed at autopsy and transported to the University of California San Diego. There the brain was prepared for frozen sectioning and cut into 2401, 70 µm coronal slices. While preliminary analyses of the brain sections have been reported, a comprehensive microscopic neuroanatomical analysis of the state of H.M.'s brain at the time of his death has not yet been published. The brain tissue and slides were subsequently moved to the University of California Davis and the slides digitized at high resolution. Initial stages of producing a website for the public viewing of the images were also carried out. Recently, the slides, digital images, and tissue have been transferred to Boston University for permanent archiving. A new steering committee has been established and plans are in place for completion of a freely accessible H.M. website. Research publications on the microscopic anatomy and neuropathology of H.M.'s brain at the time of his death are also planned. We write this commentary to provide the hippocampus and memory neuroscience communities with a brief summary of what has transpired following H.M.'s death and outline plans for future publications and a tissue archive.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

A neural stem cell paradigm of pediatric hydrocephalus.

Pediatric hydrocephalus, the leading reason for brain surgery in children, is characterized by enlargement of the cerebral ventricles classically attributed to cerebrospinal fluid (CSF) overaccumulation. Neurosurgical shunting to reduce CSF volume is the default treatment that intends to reinstate normal CSF homeostasis, yet neurodevelopmental disability often persists in hydrocephalic children despite optimal surgical management. Here, we discuss recent human genetic and animal model studies that are shifting the view of pediatric hydrocephalus from an impaired fluid plumbing model to a new paradigm of dysregulated neural stem cell (NSC) fate. NSCs are neuroprogenitor cells that comprise the germinal neuroepithelium lining the prenatal brain ventricles. We propose that heterogenous defects in the development of these cells converge to disrupt cerebrocortical morphogenesis, leading to abnormal brain-CSF biomechanical interactions that facilitate passive pooling of CSF and secondary ventricular distention. A significant subset of pediatric hydrocephalus may thus in fact be due to a developmental brain malformation leading to secondary enlargement of the ventricles rather than a primary defect of CSF circulation. If hydrocephalus is indeed a neuroradiographic presentation of an inborn brain defect, it suggests the need to focus on optimizing neurodevelopment, rather than CSF diversion, as the primary treatment strategy for these children.

Primary cilia safeguard cortical neurons in neonatal mouse forebrain from environmental stress-induced dendritic degeneration.

The developing brain is under the risk of exposure to a multitude of environmental stressors. While perinatal exposure to excessive levels of environmental stress is responsible for a wide spectrum of neurological and psychiatric conditions, the developing brain is equipped with intrinsic cell protection, the mechanisms of which remain unknown. Here we show, using neonatal mouse as a model system, that primary cilia, hair-like protrusions from the neuronal cell body, play an essential role in protecting immature neurons from the negative impacts of exposure to environmental stress. More specifically, we found that primary cilia prevent the degeneration of dendritic arbors upon exposure to alcohol and ketamine, two major cell stressors, by activating cilia-localized insulin-like growth factor 1 receptor and downstream Akt signaling. We also found that activation of this pathway inhibits Caspase-3 activation and caspase-mediated cleavage/fragmentation of cytoskeletal proteins in stress-exposed neurons. These results indicate that primary cilia play an integral role in mitigating adverse impacts of environmental stressors such as drugs on perinatal brain development.

An assessment of the existence of adult neurogenesis in humans and value of its rodent models for neuropsychiatric diseases.

In sub-mammalian vertebrates like fishes, amphibians, and reptiles, new neurons are produced during the entire lifespan. This capacity diminishes considerably in birds and even more in mammals where it persists only in the olfactory system and hippocampal dentate gyrus. Adult neurogenesis declines even more drastically in nonhuman primates and recent evidence shows that this is basically extinct in humans. Why should such seemingly useful capacity diminish during primate evolution? It has been proposed that this occurs because of the need to retain acquired complex knowledge in stable populations of neurons and their synaptic connections during many decades of human life. In this review, we will assess critically the claim of significant adult neurogenesis in humans and show how current evidence strongly indicates that humans lack this trait. In addition, we will discuss the allegation of many rodent studies that adult neurogenesis is involved in psychiatric diseases and that it is a potential mechanism for human neuron replacement and regeneration. We argue that these reports, which usually neglect significant structural and functional species-specific differences, mislead the general population into believing that there might be a cure for a variety of neuropsychiatric diseases as well as stroke and brain trauma by genesis of new neurons and their incorporation into existing synaptic circuitry.

Disorder of Golgi Apparatus Precedes Anoxia-Induced Pathology of Mitochondria.

Mitochondrial malfunction and morphologic disorganization have been observed in brain cells as part of complex pathological changes. However, it is unclear what may be the role of mitochondria in the initiation of pathologic processes or if mitochondrial disorders are consequences of earlier events. We analyzed the morphologic reorganization of organelles in an embryonic mouse brain during acute anoxia using an immunohistochemical identification of the disordered mitochondria, followed by electron microscopic three-dimensional (3D) reconstruction. We found swelling of the mitochondrial matrix after 3 h anoxia and probable dissociation of mitochondrial stomatin-like protein 2 (SLP2)-containing complexes after 4.5 h anoxia in the neocortex, hippocampus, and lateral ganglionic eminence. Surprisingly, deformation of the Golgi apparatus (GA) was detected already after 1 h of anoxia, when the mitochondria and other organelles still had a normal ultrastructure. The disordered GA showed concentrical swirling of the cisternae and formed spherical onion-like structures with the trans-cisterna in the center of the sphere. Such disturbance of the Golgi architecture likely interferes with its function for post-translational protein modification and secretory trafficking. Thus, the GA in embryonic mouse brain cells may be more vulnerable to anoxic conditions than the other organelles, including mitochondria.

The Role of Galanin in Cerebellar Granule Cell Migration in the Early Postnatal Mouse during Normal Development and after Injury.

Galanin, one of the most inducible neuropeptides, is widely present in developing brains, and its expression is altered by pathologic events (e.g., epilepsy, ischemia, and axotomy). The roles of galanin in brain development under both normal and pathologic conditions have been hypothesized, but the question of how galanin is involved in fetal and early postnatal brain development remains largely unanswered. In this study, using granule cell migration in the cerebellum of early postnatal mice (both sexes) as a model system, we examined the role of galanin in neuronal cell migration during normal development and after brain injury. Here we show that, during normal development, endogenous galanin participates in accelerating granule cell migration via altering the Ca2+ and cAMP signaling pathways. Upon brain injury induced by the application of cold insults, galanin levels decrease at the lesion sites, but increase in the surroundings of lesion sites. Granule cells exhibit the following corresponding changes in migration: (1) slowing down migration at the lesion sites; and (2) accelerating migration in the surroundings of lesion sites. Experimental manipulations of galanin signaling reduce the lesion site-specific changes in granule cell migration, indicating that galanin plays a role in such deficits in neuronal cell migration. The present study suggests that manipulating galanin signaling may be a potential therapeutic target for acutely injured brains during development.SIGNIFICANCE STATEMENT Deficits in neuronal cell migration caused by brain injury result in abnormal development of cortical layers, but the underlying mechanisms remain to be determined. Here, we report that on brain injury, endogenous levels of galanin, a neuropeptide, are altered in a lesion site-specific manner, decreasing at the lesion sites but increasing in the surroundings of lesion sites. The changes in galanin levels positively correlate with the migration rate of immature neurons. Manipulations of galanin signaling ameliorate the effects of injury on neuronal migration and cortical layer development. These results shed a light on galanin as a potential therapeutic target for acutely injured brains during development.

Gliogenesis in the outer subventricular zone promotes enlargement and gyrification of the primate cerebrum.

The primate cerebrum is characterized by a large expansion of cortical surface area, the formation of convolutions, and extraordinarily voluminous subcortical white matter. It was recently proposed that this expansion is primarily driven by increased production of superficial neurons in the dramatically enlarged outer subventricular zone (oSVZ). Here, we examined the development of the parietal cerebrum in macaque monkey and found that, indeed, the oSVZ initially adds neurons to the superficial layers II and III, increasing their thickness. However, as the oSVZ grows in size, its output changes to production of astrocytes and oligodendrocytes, which in primates outnumber cerebral neurons by a factor of three. After the completion of neurogenesis around embryonic day (E) 90, when the cerebrum is still lissencephalic, the oSVZ enlarges and contains Pax6+/Hopx+ outer (basal) radial glial cells producing astrocytes and oligodendrocytes until after E125. Our data indicate that oSVZ gliogenesis, rather than neurogenesis, correlates with rapid enlargement of the cerebrum and development of convolutions, which occur concomitantly with the formation of cortical connections via the underlying white matter, in addition to neuronal growth, elaboration of dendrites, and amplification of neuropil in the cortex, which are primary factors in the formation of cerebral convolutions in primates.

Radial Glial Cells: New Views on Old Questions.

Radial glial cells (RGC) are at the center of brain development in vertebrates, acting as progenitors for neurons and macroglia (oligodendrocytes and astrocytes) and as guides for migration of neurons from the ventricular surface to their final positions in the brain. These cells originate from neuroepithelial cells (NEC) from which they inherit their epithelial features and polarized morphology, with processes extending from the ventricular to the pial surface of the embryonic cerebrum. We have learnt a great deal since the first descriptions of these cells at the end of the nineteenth century. However, there are still questions regarding how and when NEC transform into RGC or about the function of intermediate filaments such as glial fibrillary acidic protein (GFAP) in RGCs and their dynamics during neurogenesis. For example, it is not clear why RGCs in primates, including humans, express GFAP at the onset of cortical neurogenesis while in rodents it is expressed when it is essentially complete. Based on an ultrastructural analysis of GFAP expression and cell morphology of dividing progenitors in the developing neocortex of the macaque monkey, we show that RGCs become the main progenitor in the developing cerebrum by the start of neurogenesis, as all dividing cells show glial features such as GFAP expression and lack of tight junctions. Also, our data suggest that RGCs retract their apical process during mitosis. We discuss our findings in the context of the role and molecular characteristics of RGCs in the vertebrate brain, their differences with NECs and their dynamic behavior during the process of neurogenesis.

Hypothalamic POMC neurons promote cannabinoid-induced feeding.

Hypothalamic pro-opiomelanocortin (POMC) neurons promote satiety. Cannabinoid receptor 1 (CB1R) is critical for the central regulation of food intake. Here we test whether CB1R-controlled feeding in sated mice is paralleled by decreased activity of POMC neurons. We show that chemical promotion of CB1R activity increases feeding, and notably, CB1R activation also promotes neuronal activity of POMC cells. This paradoxical increase in POMC activity was crucial for CB1R-induced feeding, because designer-receptors-exclusively-activated-by-designer-drugs (DREADD)-mediated inhibition of POMC neurons diminishes, whereas DREADD-mediated activation of POMC neurons enhances CB1R-driven feeding. The Pomc gene encodes both the anorexigenic peptide α-melanocyte-stimulating hormone, and the opioid peptide ß-endorphin. CB1R activation selectively increases ß-endorphin but not α-melanocyte-stimulating hormone release in the hypothalamus, and systemic or hypothalamic administration of the opioid receptor antagonist naloxone blocks acute CB1R-induced feeding. These processes involve mitochondrial adaptations that, when blocked, abolish CB1R-induced cellular responses and feeding. Together, these results uncover a previously unsuspected role of POMC neurons in the promotion of feeding by cannabinoids.

Metabolic regulation and glucose sensitivity of cortical radial glial cells.

The primary stem cells of the cerebral cortex are the radial glial cells (RGCs), and disturbances in their operation lead to myriad brain disorders in all mammals from mice to humans. Here, we found in mice that maternal gestational obesity and hyperglycemia can impair the maturation of RGC fibers and delay cortical neurogenesis. To investigate potential mechanisms, we used optogenetic live-imaging approaches in embryonic cortical slices. We found that Ca2+ signaling regulates mitochondrial transport and is crucial for metabolic support in RGC fibers. Cyclic intracellular Ca2+ discharge from localized RGC fiber segments detains passing mitochondria and ensures their proper distribution and enrichment at specific sites such as endfeet. Impairment of mitochondrial function caused an acute loss of Ca2+ signaling, while hyperglycemia decreased Ca2+ activity and impaired mitochondrial transport, leading to degradation of the RGC scaffold. Our findings uncover a physiological mechanism indicating pathways by which gestational metabolic disturbances can interfere with brain development.

Age-related calcium dysregulation linked with tau pathology and impaired cognition in non-human primates.

INTRODUCTION: The etiology of sporadic Alzheimer's disease (AD) requires non-genetically modified animal models. METHODS: The relationship of tau phosphorylation to calcium-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) dysregulation was analyzed in aging rhesus macaque dorsolateral prefrontal cortex (dlPFC) and rat primary cortical neurons using biochemistry and immuno-electron microscopy. The influence of calcium leak from ryanodine receptors (RyRs) on neuronal firing and cognitive performance was examined in aged macaques. RESULTS: Aged monkeys naturally develop hyperphosphorylated tau, including AD biomarkers (AT8 (pS202/pT205) and pT217) and early tau pathology markers (pS214 and pS356) that correlated with evidence of increased calcium leak (pS2808-RyR2). Calcium also regulated early tau phosphorylation in vitro. Age-related reductions in the calcium-binding protein, calbindin, and in phosphodiesterase PDE4D were seen within dlPFC pyramidal cell dendrites. Blocking RyRs with S107 improved neuronal firing and cognitive performance in aged macaques. DISCUSSION: Dysregulated calcium signaling confers risk for tau pathology and provides a potential therapeutic target.

Human-specific regulation of MeCP2 levels in fetal brains by microRNA miR-483-5p.

Proper neurological function in humans requires precise control of levels of the epigenetic regulator methyl CpG-binding protein 2 (MeCP2). MeCP2 protein levels are low in fetal brains, where the predominant MECP2 transcripts have an unusually long 3' untranslated region (UTR). Here, we show that miR-483-5p, an intragenic microRNA of the imprinted IGF2, regulates MeCP2 levels through a human-specific binding site in the MECP2 long 3' UTR. We demonstrate the inverse correlation of miR-483-5p and MeCP2 levels in developing human brains and fibroblasts from Beckwith-Wiedemann syndrome patients. Importantly, expression of miR-483-5p rescues abnormal dendritic spine phenotype of neurons overexpressing human MeCP2. In addition, miR-483-5p modulates the levels of proteins of the MeCP2-interacting corepressor complexes, including HDAC4 and TBL1X. These data provide insight into the role of miR-483-5p in regulating the levels of MeCP2 and interacting proteins during human fetal development.

Noradrenergic α1-Adrenoceptor Actions in the Primate Dorsolateral Prefrontal Cortex.

Noradrenergic (NE) α1-adrenoceptors (α1-ARs) contribute to arousal mechanisms and play an important role in therapeutic medications such as those for the treatment of posttraumatic stress disorder (PTSD). However, little is known about how α1-AR stimulation influences neuronal firing in the dorsolateral prefrontal cortex (dlPFC), a newly evolved region that is dysfunctional in PTSD and other mental illnesses. The current study examined the effects of α1-AR manipulation on neuronal firing in dlPFC of rhesus monkeys performing a visuospatial working memory task, focusing on the "delay cells" that maintain spatially tuned information across the delay period. Iontophoresis of the α1-AR antagonist HEAT (2-{[ß-(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone) had mixed effects, reducing firing in a majority of neurons but having nonsignificant excitatory effects or no effect in remaining delay cells. These data suggest that endogenous NE has excitatory effects in some delay cells under basal conditions. In contrast, the α1-AR agonists phenylephrine and cirazoline suppressed delay cell firing and this was blocked by coadministration of HEAT. These results indicate an inverted-U dose response for α1-AR actions, with mixed excitatory actions under basal conditions and suppressed firing with high levels of α1-AR stimulation such as with stress exposure. Immunoelectron microscopy revealed α1-AR expression presynaptically in axons and axon terminals and postsynaptically in spines, dendrites, and astrocytes. It is possible that α1-AR excitatory effects arise from presynaptic excitation of glutamate release, whereas postsynaptic actions suppress firing through calcium-protein kinase C opening of potassium channels on spines. The latter may predominate under stressful conditions, leading to loss of dlPFC regulation during uncontrollable stress.SIGNIFICANCE STATEMENT Noradrenergic stimulation of α1-adrenoceptors (α1-ARs) is implicated in posttraumatic stress disorder (PTSD) and other mental disorders that involve dysfunction of the prefrontal cortex, a brain region that provides top-down control. However, the location and contribution of α1-ARs to prefrontal cortical physiology in primates has received little attention. This study found that α1-ARs are located near prefrontal synapses and that α1-AR stimulation has mixed effects under basal conditions. However, high levels of α1-AR stimulation, as occur with stress, suppress neuronal firing. These findings help to explain why we lose top-down control under conditions of uncontrollable stress when there are high levels of noradrenergic release in brain and why blocking α1-AR, such as with prazosin, may be helpful in the treatment of PTSD.

Classical complement cascade initiating C1q protein within neurons in the aged rhesus macaque dorsolateral prefrontal cortex.

BACKGROUND: Cognitive impairment in schizophrenia, aging, and Alzheimer's disease is associated with spine and synapse loss from the dorsolateral prefrontal cortex (dlPFC) layer III. Complement cascade signaling is critical in driving spine loss and disease pathogenesis. Complement signaling is initiated by C1q, which tags synapses for elimination. C1q is thought to be expressed predominately by microglia, but its expression in primate dlPFC has never been examined. The current study assayed C1q levels in aging primate dlPFC and rat medial PFC (mPFC) and used immunoelectron microscopy (immunoEM), immunoblotting, and co-immunoprecipitation (co-IP) to reveal the precise anatomical distribution and interactions of C1q. METHODS: Age-related changes in C1q levels in rhesus macaque dlPFC and rat mPFC were examined using immunoblotting. High-spatial resolution immunoEM was used to interrogate the subcellular localization of C1q in aged macaque layer III dlPFC and aged rat layer III mPFC. co-IP techniques quantified protein-protein interactions for C1q and proteins associated with excitatory and inhibitory synapses in macaque dlPFC. RESULTS: C1q levels were markedly increased in the aged macaque dlPFC. Ultrastructural localization found the expected C1q localization in glia, including those ensheathing synapses, but also revealed extensive localization within neurons. C1q was found near synapses, within terminals and in spines, but was also observed in dendrites, often near abnormal mitochondria. Similar analyses in aging rat mPFC corroborated the findings in rhesus macaques. C1q protein increasingly associated with PSD95 with age in macaque, consistent with its synaptic localization as evidenced by EM. CONCLUSIONS: These findings reveal novel, intra-neuronal distribution patterns for C1q in the aging primate cortex, including evidence of C1q in dendrites. They suggest that age-related changes in the dlPFC may increase C1q expression and synaptic tagging for glial phagocytosis, a possible mechanism for age-related degeneration.