Imaging neural circuit pathology of autism spectrum disorders: autism-associated genes, animal models and the application of in vivo two-photon imaging.

Recent advances in human genetics identified genetic variants involved in causing autism spectrum disorders (ASDs). Mouse models that mimic mutations found in patients with ASD exhibit behavioral phenotypes consistent with ASD symptoms. These mouse models suggest critical biological factors of ASD etiology. Another important implication of ASD genetics is the enrichment of ASD risk genes in molecules involved in developing synapses and regulating neural circuit function. Sophisticated in vivo imaging technologies applied to ASD mouse models identify common synaptic impairments in the neocortex, with genetic-mutation-specific defects in local neural circuits. In this article, we review synapse- and circuit-level phenotypes identified by in vivo two-photon imaging in multiple mouse models of ASD and discuss the contributions of altered synapse properties and neural circuit activity to ASD pathogenesis.

Neurodegenerative processes accelerated by protein malnutrition and decelerated by essential amino acids in a tauopathy mouse model.

Protein malnutrition is epidemiologically suggested as a potential risk factor for senile dementia, although molecular mechanisms linking dietary proteins and amino acids to neurodegeneration remain unknown. Here, we show that a low-protein diet resulted in down-regulated expression of synaptic components and a modest acceleration of brain atrophy in mice modeling neurodegenerative tauopathies. Notably, these abnormal phenotypes were robustly rescued by the administration of seven selected essential amino acids. The up-regulation of inflammation-associated gene expression and progressive brain atrophy in the tauopathy model were profoundly suppressed by treatment with these essential amino acids without modifications of tau depositions. Moreover, the levels of kynurenine, an initiator of a pathway inducing neuroinflammatory gliosis and neurotoxicity in the brain, were lowered by treatment through inhibition of kynurenine uptake in the brain. Our findings highlight the importance of specific amino acids as systemic mediators of brain homeostasis against neurodegenerative processes.

Selective Disruption of Inhibitory Synapses Leading to Neuronal Hyperexcitability at an Early Stage of Tau Pathogenesis in a Mouse Model.

Synaptic dysfunction provoking dysregulated cortical neural circuits is currently hypothesized as a key pathophysiological process underlying clinical manifestations in Alzheimer's disease and related neurodegenerative tauopathies. Here, we conducted PET along with postmortem assays to investigate time course changes of excitatory and inhibitory synaptic constituents in an rTg4510 mouse model of tauopathy, which develops tau pathologies leading to noticeable brain atrophy at 5-6 months of age. Both male and female mice were analyzed in this study. We observed that radiosignals derived from [11C]flumazenil, a tracer for benzodiazepine receptor, in rTg4510 mice were significantly lower than the levels in nontransgenic littermates at 2-3 months of age. In contrast, retentions of (E)-[11C]ABP688, a tracer for mGluR5, were unaltered relative to controls at 2 months of age but then gradually declined with aging in parallel with progressive brain atrophy. Biochemical and immunohistochemical assessment of postmortem brain tissues demonstrated that inhibitory, but not excitatory, synaptic constituents selectively diminished without overt loss of somas of GABAergic interneurons in the neocortex and hippocampus of rTg4510 mice at 2 months of age, which was concurrent with enhanced immunoreactivity of cFos, a well-characterized immediate early gene, suggesting that impaired inhibitory neurotransmission may cause hyperexcitability of cortical circuits. Our findings indicate that tau-induced disruption of the inhibitory synapse may be a critical trigger of progressive neurodegeneration, resulting in massive neuronal loss, and PET assessments of inhibitory versus excitatory synapses potentially offer in vivo indices for hyperexcitability and excitotoxicity early in the etiologic pathway of neurodegenerative tauopathies.SIGNIFICANCE STATEMENT In this study, we examined the in vivo status of excitatory and inhibitory synapses in the brain of the rTg4510 tauopathy mouse model by PET imaging with (E)-[11C]ABP688 and [11C]flumazenil, respectively. We identified inhibitory synapse as being significantly dysregulated before brain atrophy at 2 months of age, while excitatory synapse stayed relatively intact at this stage. In line with this observation, postmortem assessment of brain tissues demonstrated selective attenuation of inhibitory synaptic constituents accompanied by the upregulation of cFos before the formation of tau pathology in the forebrain at young ages. Our findings indicate that selective degeneration of inhibitory synapse with hyperexcitability in the cortical circuit constitutes the critical early pathophysiology of tauopathy.

Inverse synaptic tagging: An inactive synapse-specific mechanism to capture activity-induced Arc/arg3.1 and to locally regulate spatial distribution of synaptic weights.

Long-lasting forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) are fundamental cellular mechanisms underlying learning and memory. The synaptic tagging and capture (STC) hypothesis has provided a theoretical framework on how products of activity-dependent genes may interact with potentiated synapses to facilitate and maintain such long-lasting synaptic plasticity. Although Arc/arg3.1 was initially assumed to participate in STC processes during LTP, accumulating evidence indicated that Arc/arg3.1 might rather contribute in weakening of synaptic weights than in their strengthening. In particular, analyses of Arc/Arg3.1 protein dynamics and function in the dendrites after plasticity-inducing stimuli have revealed a new type of inactivity-dependent redistribution of synaptic weights, termed "inverse synaptic tagging". The original synaptic tagging and inverse synaptic tagging likely co-exist and are mutually non-exclusive mechanisms, which together may help orchestrate the redistribution of synaptic weights and promote the enhancement and maintenance of their contrast between potentiated and non-potentiated synapses during the late phase of long-term synaptic plasticity. In this review, we describe the inverse synaptic tagging mechanism that controls synaptic dynamics of Arc/Arg3.1, an immediate early gene product which is captured and preferentially targeted to non-potentiated synapses, and discuss its impact on neuronal circuit refinement and cognitive function.

An intracellular domain with a novel sequence regulates cell surface expression and synaptic clustering of leucine-rich repeat transmembrane proteins in hippocampal neurons.

Leucine-rich repeat transmembrane proteins (LRRTMs) are single-spanning transmembrane proteins that belong to the family of synaptically localized adhesion molecules that play various roles in the formation, maturation, and function of synapses. LRRTMs are highly localized in the post-synaptic density; however, the mechanisms and significance of LRRTM synaptic clustering remain unclear. Here, we focus on the intracellular domain of LRRTMs and investigate its role in cell surface expression and synaptic clustering. The deletion of 55-56 residues in the cytoplasmic tail caused significantly reduced synaptic clustering of LRRTM1-4 in rat hippocampal neurons, whereas it simultaneously resulted in augmented LRRTM1-2 cell surface expression. A series of deletions and further single amino acid substitutions in the intracellular domain of LRRTM2 demonstrated that a previously uncharacterized sequence at the region of -16 to -13 from the C-terminus was responsible for efficient synaptic clustering and proper cell surface trafficking of LRRTMs. Furthermore, the clustering-deficient LRRTM2 mutant lost the ability to promote the accumulation of post-synaptic density protein-95 (PSD-95). These results suggest that trafficking to the cell surface and synaptic clustering of LRRTMs are regulated by a specific mechanism through this novel sequence in the intracellular domain that underlies post-synaptic molecular assembly and maturation. Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules promoting synapse formation. LRRTMs are highly localized in the postsynaptic density. We report amino acid sequence YxxC in the intracellular domain of LRRTMs is responsible for the postsynaptic localization of LRRTMs. This novel amino acid sequence of LRRTMs facilitates synapse maturation. We propose this regulated synaptic clustering of LRRTMs by the intracellular domain presents a novel molecular mechanism of synapse maturation.

Role of Immediate-Early Genes in Synaptic Plasticity and Neuronal Ensembles Underlying the Memory Trace.

In the brain, neuronal gene expression is dynamically changed in response to neuronal activity. In particular, the expression of immediate-early genes (IEGs) such as egr-1, c-fos, and Arc is rapidly and selectively upregulated in subsets of neurons in specific brain regions associated with learning and memory formation. IEG expression has therefore been widely used as a molecular marker for neuronal populations that undergo plastic changes underlying formation of long-term memory. In recent years, optogenetic and pharmacogenetic studies of neurons expressing c-fos or Arc have revealed that, during learning, IEG-positive neurons encode and store information that is required for memory recall, suggesting that they may be involved in formation of the memory trace. However, despite accumulating evidence for the role of IEGs in synaptic plasticity, the molecular and cellular mechanisms associated with this process remain unclear. In this review, we first summarize recent literature concerning the role of IEG-expressing neuronal ensembles in organizing the memory trace. We then focus on the physiological significance of IEGs, especially Arc, in synaptic plasticity, and describe our hypotheses about the importance of Arc expression in various types of input-specific circuit reorganization. Finally, we offer perspectives on Arc function that would unveil the role of IEG-expressing neurons in the formation of memory traces in the hippocampus and other brain areas.

Ligand binding of PDZ domains has various roles in the synaptic clustering of SAP102 and PSD-95.

Synapse-associated protein 102 (SAP102) and postsynaptic density-95 (PSD-95) bind to NMDA receptors through PDZ domains and cluster at excitatory postsynaptic sites called postsynaptic densities (PSD). We previously reported that PSD-95 containing mutated PDZ domains incapable of ligand binding clustered at synaptic sites with reduced efficiency. Here, we compared the synaptic clustering of the same series of full-length SAP102 mutants in hippocampal neurons. Unexpectedly, ligand-binding deficient mutant SAP102 showed more efficient synaptic localization than wild-type SAP102. Further, when SAP102-PDZ mutants were co-expressed with either the GluN2A or GluN2B NMDA receptor subunit, both subunits showed decreased synaptic clustering, although the mutants were efficiently targeted to the synapses. This finding suggests that direct binding of NMDA receptors with SAP102 is involved in the efficient targeting of NMDA receptors to the synapses, whereas ligand binding of the PDZ domains is not essential for the synaptic clustering of SAP102.

Triple N-glycosylation in the long S5-P loop regulates the activation and trafficking of the Kv12.2 potassium channel.

Mammalian voltage-dependent potassium (Kv) channels regulate the excitability of nerve and muscle cells. Kv12.2 features the longest S5-P loop among all known mammalian Kv channels with the most N-linked glycosylation sites (three sites). Despite its unique structural features, Kv12.2 is not well characterized. Because glycosylation plays important roles in the folding, trafficking, and function of various Kv channels, we focused on the N-glycosylation of Kv12.2. We show that Kv12.2 is N-glycosylated in Chinese hamster ovary (CHO) cells and in cultured neurons as well as in the mouse brain. As an effect of N-glycosylation on the function of Kv12.2, we demonstrate that removal of sugar chains causes a depolarizing shift in the steady-state activation without a significant reduction in current amplitude. Unlike the previously reported shift for Shaker-type Kv channels, this shift does not appear to be due to negatively charged sialic acid residues in the sugar chains. We next examined the trafficking in CHO cells to address whether the unglycosylated Kv12.2 channels are utilized in vivo. Although double mutants, retaining only one glycosylation site, are trafficked to the surface of CHO cells irrespective of the position of the glycosylated site, unglycosylated channels are not trafficked to the cell surface. Furthermore, we could not detect unglycosylated channels in the mouse brain. Our data suggest that only glycosylated Kv12.2 channels show proper voltage dependence and are utilized in vivo.