Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca2+ homeostasis and network synchrony via PMCA2/ATP2B2.

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony by a shed ectodomain.

cAMP Signaling-Mediated Phosphorylation of Diacylglycerol Lipase α Regulates Interaction With Ankyrin-G and Dendritic Spine Morphology.

BACKGROUND: Diacylglycerol lipase α (DAGLα), a major biosynthetic enzyme for endogenous cannabinoid signaling, has emerged as a risk gene in multiple psychiatric disorders. However, its role in the regulation of dendritic spine plasticity is unclear. METHODS: DAGLα wild-type or point mutants were overexpressed in primary cortical neurons or human embryonic kidney 293T cells. The effects of mutated variants on interaction, dendritic spine morphology, and dynamics were examined by proximity ligation assay or fluorescence recovery after photobleaching. Behavioral tests and immunohistochemistry were performed with ankyrin-G conditional knockout and wild-type male mice. RESULTS: DAGLα modulated dendritic spine size and density, but the effects of changes in its protein level versus enzymatic activity were different, implicating either a 2-arachidonoylglycerol (2-AG)-dependent or -independent mechanism. The 2-AG-independent effects were mediated by the interaction of DAGLα with ankyrin-G, a multifunctional scaffold protein implicated in psychiatric disorders. Using superresolution microscopy, we observed that they colocalized in distinct nanodomains, which correlated with spine size. In situ proximity ligation assay combined with structured illumination microscopy revealed that DAGLα phosphorylation upon forskolin treatment enhanced the interaction with ankyrin-G in spines, leading to increased spine size and decreased DAGLα surface diffusion. Ankyrin-G conditional knockout mice showed significantly decreased DAGLα-positive neurons in the forebrain. In mice, ankyrin-G was required for forskolin-dependent reversal of depression-related behavior. CONCLUSIONS: Taken together, ANK3 and DAGLA, both neuropsychiatric disorder genes, interact in a complex to regulate spine morphology. These data reveal novel synaptic signaling mechanisms and potential therapeutic avenues.

Rapid 3D Enhanced Resolution Microscopy Reveals Diversity in Dendritic Spinule Dynamics, Regulation, and Function.

Dendritic spinules are thin protrusions, formed by neuronal spines, not adequately resolved by diffraction-limited light microscopy, which has limited our understanding of their behavior. Here we performed rapid structured illumination microscopy and enhanced resolution confocal microscopy to study spatiotemporal spinule dynamics in cortical pyramidal neurons. Spinules recurred at the same locations on mushroom spine heads. Most were short-lived, dynamic, exploratory, and originated near simple PSDs, whereas a subset was long-lived, elongated, and associated with complex PSDs. These subtypes were differentially regulated by Ca2+ transients. Furthermore, the postsynaptic Rac1-GEF kalirin-7 regulated spinule formation, elongation, and recurrence. Long-lived spinules often contained PSD fragments, contacted distal presynaptic terminals, and formed secondary synapses. NMDAR activation increased spinule number, length, and contact with distal presynaptic elements. Spinule subsets, dynamics, and recurrence were validated in cortical neurons of acute brain slices. Thus, we identified unique properties, regulatory mechanisms, and functions of spinule subtypes, supporting roles in neuronal connectivity.

A novel role for the late-onset Alzheimer's disease (LOAD)-associated protein Bin1 in regulating postsynaptic trafficking and glutamatergic signaling.

Postsynaptic trafficking plays a key role in regulating synapse structure and function. While spiny excitatory synapses can be stable throughout adult life, their morphology and function is impaired in Alzheimer's disease (AD). However, little is known about how AD risk genes impact synaptic function. Here we used structured superresolution illumination microscopy (SIM) to study the late-onset Alzheimer's disease (LOAD) risk factor BIN1, and show that this protein is abundant in postsynaptic compartments, including spines. While postsynaptic Bin1 shows colocalization with clathrin, a major endocytic protein, it also colocalizes with the small GTPases Rab11 and Arf6, components of the exocytic pathway. Bin1 participates in protein complexes with Arf6 and GluA1, and manipulations of Bin1 lead to changes in spine morphology, AMPA receptor surface expression and trafficking, and AMPA receptor-mediated synaptic transmission. Our data provide new insights into the mesoscale architecture of postsynaptic trafficking compartments and their regulation by a major LOAD risk factor.

Usp9X Controls Ankyrin-Repeat Domain Protein Homeostasis during Dendritic Spine Development.

Variants in the ANK3 gene encoding ankyrin-G are associated with neurodevelopmental disorders, including intellectual disability, autism, schizophrenia, and bipolar disorder. However, no upstream regulators of ankyrin-G at synapses are known. Here, we show that ankyrin-G interacts with Usp9X, a neurodevelopmental-disorder-associated deubiquitinase (DUB). Usp9X phosphorylation enhances their interaction, decreases ankyrin-G polyubiquitination, and stabilizes ankyrin-G to maintain dendritic spine development. In forebrain-specific Usp9X knockout mice (Usp9X-/Y), ankyrin-G as well as multiple ankyrin-repeat domain (ANKRD)-containing proteins are transiently reduced at 2 but recovered at 12 weeks postnatally. However, reduced cortical spine density in knockouts persists into adulthood. Usp9X-/Y mice display increase of ankyrin-G ubiquitination and aggregation and hyperactivity. USP9X mutations in patients with intellectual disability and autism ablate its catalytic activity or ankyrin-G interaction. Our data reveal a DUB-dependent mechanism of ANKRD protein homeostasis, the impairment of which only transiently affects ANKRD protein levels but leads to persistent neuronal, behavioral, and clinical abnormalities.

CNTNAP2 stabilizes interneuron dendritic arbors through CASK.

Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree. These alterations can be replicated by acute knockdown of CNTNAP2 in mature wild-type (WT) neurons and are caused by faulty dendrite stabilization rather than outgrowth. Using structured illumination microscopy (SIM) and stimulated-emission depletion microscopy (STED), two super-resolution imaging techniques, we uncovered relationships between nanoscale CNTNAP2 protein localization and dendrite arborization patterns. Employing yeast two-hybrid screening, biochemical analysis, in situ proximity ligation assay (PLA), SIM, and phenotype rescue, we show that these effects are mediated at the membrane by the interaction of CNTNAP2's C-terminus with calcium/calmodulin-dependent serine protein kinase (CASK), another ASD/ID risk gene. Finally, we show that adult Cntnap2 KO mice have reduced interneuron dendritic length and branching in particular cortical regions, as well as decreased CASK levels in the cortical membrane fraction. Taken together, our data reveal an interneuron-specific mechanism for dendrite stabilization that may provide a cellular mechanism for inhibitory circuit dysfunction in CNTNAP2-related disorders.

Short-term modern life-like stress exacerbates Aß-pathology and synapse loss in 3xTg-AD mice.

Alzheimer's disease (AD) is a progressive neurological disorder that impairs memory and other cognitive functions in the elderly. The social and financial impacts of AD are overwhelming and are escalating exponentially as a result of population aging. Therefore, identifying AD-related risk factors and the development of more efficacious therapeutic approaches are critical to cure this neurological disorder. Current epidemiological evidence indicates that life experiences, including chronic stress, are a risk for AD. However, it is unknown if short-term stress, lasting for hours, influences the onset or progression of AD. Here, we determined the effect of short-term, multi-modal 'modern life-like' stress on AD pathogenesis and synaptic plasticity in mice bearing three AD mutations (the 3xTg-AD mouse model). We found that combined emotional and physical stress lasting 5 h severely impaired memory in wild-type mice and tended to impact it in already low-performing 3xTg-AD mice. This stress reduced the number of synapse-bearing dendritic spines in 3xTg-AD mice and increased Aß levels by augmenting AßPP processing. Thus, short-term stress simulating modern-life conditions may exacerbate cognitive deficits in preclinical AD by accelerating amyloid pathology and reducing synapse numbers. Epidemiological evidence indicates that life experiences, including chronic stress, are a risk for Alzheimer disease (AD). However, it is unknown if short stress in the range of hours influences the onset or progression of AD. Here, we determined the effect of short, multi-modal 'modern-lifelike'stress on AD pathogenesis and synaptic plasticity in mice bearing three AD mutations (the 3xTg-AD mouse model). We found that combined emotional and physical stress lasting 5 h severely impaired memory in wild-type mice and tended to impact it in already low-performing 3xTg-AD mice. This stress reduced the number of synapse-bearing dendritic spines in 3xTg-AD mice and increased Aß levels by augmenting AßPP processing. Thus, short stress simulating modern-life conditions may exacerbate cognitive deficits in preclinical AD by accelerating amyloid pathology and reducing synapse numbers.

Psychiatric risk factor ANK3/ankyrin-G nanodomains regulate the structure and function of glutamatergic synapses.

Recent evidence implicates glutamatergic synapses as key pathogenic sites in psychiatric disorders. Common and rare variants in the ANK3 gene, encoding ankyrin-G, have been associated with bipolar disorder, schizophrenia, and autism. Here we demonstrate that ankyrin-G is integral to AMPAR-mediated synaptic transmission and maintenance of spine morphology. Using superresolution microscopy we find that ankyrin-G forms distinct nanodomain structures within the spine head and neck. At these sites, it modulates mushroom spine structure and function, probably as a perisynaptic scaffold and barrier within the spine neck. Neuronal activity promotes ankyrin-G accumulation in distinct spine subdomains, where it differentially regulates NMDA receptor-dependent plasticity. These data implicate subsynaptic nanodomains containing a major psychiatric risk molecule, ankyrin-G, as having location-specific functions and open directions for basic and translational investigation of psychiatric risk molecules.

Hippocampal adaptive response following extensive neuronal loss in an inducible transgenic mouse model.

Neuronal loss is a common component of a variety of neurodegenerative disorders (including Alzheimer's, Parkinson's, and Huntington's disease) and brain traumas (stroke, epilepsy, and traumatic brain injury). One brain region that commonly exhibits neuronal loss in several neurodegenerative disorders is the hippocampus, an area of the brain critical for the formation and retrieval of memories. Long-lasting and sometimes unrecoverable deficits caused by neuronal loss present a unique challenge for clinicians and for researchers who attempt to model these traumas in animals. Can these deficits be recovered, and if so, is the brain capable of regeneration following neuronal loss? To address this significant question, we utilized the innovative CaM/Tet-DT(A) mouse model that selectively induces neuronal ablation. We found that we are able to inflict a consistent and significant lesion to the hippocampus, resulting in hippocampally-dependent behavioral deficits and a long-lasting upregulation in neurogenesis, suggesting that this process might be a critical part of hippocampal recovery. In addition, we provide novel evidence of angiogenic and vasculature changes following hippocampal neuronal loss in CaM/Tet-DTA mice. We posit that angiogenesis may be an important factor that promotes neurogenic upregulation following hippocampal neuronal loss, and both factors, angiogenesis and neurogenesis, can contribute to the adaptive response of the brain for behavioral recovery.

Synergistic Interactions between Abeta, tau, and alpha-synuclein: acceleration of neuropathology and cognitive decline.

Alzheimer's disease (AD), the most prevalent age-related neurodegenerative disorder, is characterized pathologically by the accumulation of beta-amyloid (Abeta) plaques and tau-laden neurofibrillary tangles. Interestingly, up to 50% of AD cases exhibit a third prevalent neuropathology: the aggregation of alpha-synuclein into Lewy bodies. Importantly, the presence of Lewy body pathology in AD is associated with a more aggressive disease course and accelerated cognitive dysfunction. Thus, Abeta, tau, and alpha-synuclein may interact synergistically to promote the accumulation of each other. In this study, we used a genetic approach to generate a model that exhibits the combined pathologies of AD and dementia with Lewy bodies (DLB). To achieve this goal, we introduced a mutant human alpha-synuclein transgene into 3xTg-AD mice. As occurs in human disease, transgenic mice that develop both DLB and AD pathologies (DLB-AD mice) exhibit accelerated cognitive decline associated with a dramatic enhancement of Abeta, tau, and alpha-synuclein pathologies. Our findings also provide additional evidence that the accumulation of alpha-synuclein alone can significantly disrupt cognition. Together, our data support the notion that Abeta, tau, and alpha-synuclein interact in vivo to promote the aggregation and accumulation of each other and accelerate cognitive dysfunction.

Better than average: alternatives to statistical summary representations for rapid judgments of average size.

People can perceive the individual features of an object by focusing attention on it and binding the features together at a location. Some perceptual processing can occur without focusing attention on each object, though; people may even be able to extract summary information about the sizes of all the objects in a display, essentially computing the mean size at a glance. Evidence that people can judge the mean size of an array efficiently and accurately has been used to support the strong claim that people use a global, parallel process to extract a statistical summary of the average size of the objects in the display. Such claims are based both on the accuracy of performance and on the supposition that performance exceeds what would be possible with serial, focused attention. However, these studies typically have not examined the limits of performance with focused-attention strategies. Through experiments and simulations, we show that existing evidence for mean size perception can be explained through various focused-attention strategies, without appealing to a new mechanism of average size perception. Although our evidence does not eliminate the possibility that people do perceive the average size of all the objects in a display, it suggests that simpler mechanisms can accommodate the existing data.