Characterization of 18F-PM-PBB3 (18F-APN-1607) Uptake in the rTg4510 Mouse Model of Tauopathy.

Misfolding, aggregation, and cerebral accumulation of tau deposits are hallmark features of Alzheimer's disease. Positron emission tomography study of tau can facilitate the development of anti-tau treatment. Here, we investigated a novel tau tracer 18F-PM-PBB3 (18F-APN-1607) in a mouse model of tauopathy. Dynamic PET scans were collected in groups of rTg4510 transgenic mice at 2-11 months of age. Associations between distribution volume ratios (DVR) and standardized uptake value ratios (SUVR) with cerebellum reference were used to determine the optimal scanning time and uptake pattern for each age. Immunohistochemistry staining of neurofibrillary tangles and autoradiography study was performed for ex vivo validation. An SUVR 40-70 min was most consistently correlated with DVR and was used in further analyses. Significant increased 18F-PM-PBB3 uptake in the brain cortex was found in six-month-old mice (+28.9%, p < 0.05), and increased further in the nine-month-old group (+38.8%, p < 0.01). The trend of increased SUVR value remained evident in the hippocampus and striatum regions except for cortex where uptake becomes slightly reduced in 11-month-old animals (+37.3%, p < 0.05). Radioactivity distributions from autoradiography correlate well to the presence of human tau (HT7 antibody) and hyperphosphorylated tau (antibody AT8) from the immunohistochemistry study of the adjacent brain sections. These findings supported that the 40-70 min 18F-PM-PBB3 PET scan with SUVR measurement can detect significantly increased tau deposits in a living rTg4510 transgenic mouse models as early as six-months-old. The result exhibited promising dynamic imaging capability of this novel tau tracer, and the above image characteristics should be considered in the design of longitudinal preclinical tau image studies.

Rab18 Collaborates with Rab7 to Modulate Lysosomal and Autophagy Activities in the Nervous System: an Overlapping Mechanism for Warburg Micro Syndrome and Charcot-Marie-Tooth Neuropathy Type 2B.

Mutations in RAB18, a member of small G protein, cause Warburg micro syndrome (WARBM), whose clinical features include vision impairment, postnatal microcephaly, and lower limb spasticity. Previously, our Rab18-/- mice exhibited hind limb weakness and spasticity as well as signs of axonal degeneration in the spinal cord and lumbar spinal nerves. However, the cellular and molecular function of RAB18 and its roles in the pathogenesis of WARBM are still not fully understood. Using immunofluorescence staining and expression of Rab18 and organelle markers, we find that Rab18 associates with lysosomes and actively traffics along neurites in cultured neurons. Interestingly, Rab18-/- neurons exhibit impaired lysosomal transport. Using autophagosome marker LC3-II, we show that Rab18 dysfunction leads to aberrant autophagy activities in neurons. Electron microscopy further reveals accumulation of lipofuscin-like granules in the dorsal root ganglion of Rab18-/- mice. Surprisingly, Rab18 colocalizes, cofractionates, and coprecipitates with the lysosomal regulator Rab7, mutations of which cause Charcot-Marie-Tooth (CMT) neuropathy type 2B. Moreover, Rab7 is upregulated in Rab18-deficient neurons, suggesting a compensatory effect. Together, our results suggest that the functions of RAB18 and RAB7 in lysosomal and autophagic activities may constitute an overlapping mechanism underlying WARBM and CMT pathogenesis in the nervous system.

µ2-Dependent endocytosis of N-cadherin is regulated by ß-catenin to facilitate neurite outgrowth.

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the µ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, ß-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of ß-catenin facilitates µ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through ß-catenin-regulated µ2 binding to modulate neurite outgrowth.

Postsynaptic Y654 dephosphorylation of ß-catenin modulates presynaptic vesicle turnover through increased n-cadherin-mediated transsynaptic signaling.

Synaptic adhesion molecules, which coordinately control structural and functional changes at both sides of synapses, are important for synaptogenesis and synaptic plasticity. Because they physically form homophilic or heterophilic adhesions across synaptic junctions, these molecules can initiate transsynaptic communication in both anterograde and retrograde directions. Using optical imaging approaches, we investigated whether an increase in postsynaptic N-cadherin could correspondingly alter the function of connected presynaptic terminals. Postsynaptic expression of ß-catenin Y654F, a phosphorylation-defective form with enhanced binding to N-cadherin, is sufficient to increase postsynaptic surface levels of N-cadherin and consequently promote presynaptic reorganizations. Such reorganizations include increases in the densities of the synaptic vesicle protein, Synaptotagmin 1 and the active zone scaffold protein, Bassoon, the number of active boutons and the size of the total recycling vesicle pool. In contrast, synaptic vesicle turnover is significantly impaired, preventing the exchange of synaptic vesicles with adjacent boutons. Together, N-cadherin-mediated retrograde signaling, governed by phosphoregulation of postsynaptic ß-catenin Y654, coordinately modulates presynaptic vesicle dynamics to enhance synaptic communication in mature neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 61-74, 2017.

PRRT2 mutations lead to neuronal dysfunction and neurodevelopmental defects.

Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene cause a wide spectrum of neurological diseases, ranging from paroxysmal kinesigenic dyskinesia (PKD) to mental retardation and epilepsy. Previously, seven PKD-related PRRT2 heterozygous mutations were identified in the Taiwanese population: P91QfsX, E199X, S202HfsX, R217PfsX, R217EfsX, R240X and R308C. This study aimed to investigate the disease-causing mechanisms of these PRRT2 mutations. We first documented that Prrt2 was localized at the pre- and post-synaptic membranes with a close spatial association with SNAP25 by synaptic membrane fractionation and immunostaining of the rat neurons. Our results then revealed that the six truncating Prrt2 mutants were accumulated in the cytoplasm and thus failed to target to the cell membrane; the R308C missense mutant had significantly reduced protein expression, suggesting loss-of function effects generated by these mutations. Using in utero electroporation of shRNA into cortical neurons, we further found that knocking down Prrt2 expression in vivo resulted in a delay in neuronal migration during embryonic development and a marked decrease in synaptic density after birth. These pathologic effects and novel disease-causing mechanisms may contribute to the severe clinical symptoms in PRRT2-related diseases.

N-Cadherin Regulates Cell Migration Through a Rab5-Dependent Temporal Control of Macropinocytosis.

Macropinocytosis is a clathrin-independent endocytic pathway implicated in fluid uptake, pathogen invasion and cell migration. During collective cell migration, macropinocytosis occurs primarily at membrane ruffles arising from the leading edges of migrating cells. We report here that N-cadherin (Ncad) regulates the tempo of macropinocytosis and thereby influences wound-induced collective cell migration. Using live-cell and super-resolution imaging techniques, we observed that Ncad formed clusters at the membrane ruffles and macropinosomes. De-clustering of Ncad by an interfering antibody impaired the recruitment of Rab5-an early endosomal marker-to the macropinosomes. Moreover, we demonstrated that Ncad interacts with Rab5, and laser ablation of Ncad caused Rab5 to dissociate from the macropinosomes. Although Rab5 detached from macropinosomes upon the de-clustering of Ncad, the recruitment of late endosomal marker Rab7 occurred earlier. Consequently, both centripetal trafficking of macropinosomes and collective migration were accelerated due to de-clustering of Ncad. Thus, our results suggest that Ncad is involved in the maturation of macropinocytosis through Rab5 recruitment, linking macropinocytosis and cell migration through a novel function of Ncad.

ENU mutagenesis identifies mice modeling Warburg Micro Syndrome with sensory axon degeneration caused by a deletion in Rab18.

Mutations in the gene of RAB18, a member of Ras superfamily of small G-proteins, cause Warburg Micro Syndrome (WARBM) which is characterized by defective neurodevelopmental and ophthalmological phenotypes. Despite loss of Rab18 had been reported to induce disruption of the endoplasmic reticulum structure and neuronal cytoskeleton organization, parts of the pathogenic mechanism caused by RAB18 mutation remain unclear. From the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis library, we identified a mouse line whose Rab18 was knocked out. This Rab18(-/-) mouse exhibited stomping gait, smaller testis and eyes, mimicking several features of WARBM. Rab18(-/-) mice were obviously less sensitive to pain and touch than WT mice. Histological examinations on Rab18(-/-) mice revealed progressive axonal degeneration in the optic nerves, dorsal column of the spinal cord and sensory roots of the spinal nerves while the motor roots were spared. All the behavioral and pathological changes that resulted from abnormalities in the sensory axons were prevented by introducing an extra copy of Rab18 transgene in Rab18(-/-) mice. Our results reveal that sensory axonal degeneration is the primary cause of stomping gait and progressive weakness of the hind limbs in Rab18(-/-) mice, and optic nerve degeneration should be the major pathology of progressive optic atrophy in children with WARBM. Our results indicate that the sensory nervous system is more vulnerable to Rab18 deficiency and WARBM is not only a neurodevelopmental but also neurodegenerative disease.

Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherin-cadherin interactions are gated by extracellular Ca(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (τ = 1.17 ± 0.06 s(-1)) upon chelation of extracellular Ca(2+). A cadherin mutant with reduced adhesive activity (W2A) exhibited a faster, more substantial loss of homophilic interactions (τ = 0.86 ± 0.02 s(-1)), suggesting two types of native cadherin interactions--one that is rapidly modulated by changes in extracellular Ca(2+) and another with relatively stable adhesive activity that is Ca(2+) independent. The Ca(2+)-sensitive dynamics of cadherin interactions were transmitted to the cell interior where ß-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.

Cadherins and synaptic plasticity.

Given their trans-synaptic localization, their persistent expression at mature synapses and their distinct biochemical and adhesive properties, cadherins are uniquely poised at the synapse to mediate synaptic plasticity, the ability to change synaptic function thought to underlie learning and memory. For example recent work suggests that cadherins may recruit and stabilize AMPA receptors at the synapse via direct interactions or through complex formation, revealing cross talk between postsynaptic signaling and adhesion. Moreover, the use of small interfering RNA knockdown of cadherin, the availability of N-cadherin-deficient embryonic stem cells and the acute disruption of cadherin function with peptide application in vivo have allowed for more precise dissection of the molecular mechanisms by which cadherins function in both structural and functional plasticity.

N-cadherin, spine dynamics, and synaptic function.

Dendritic spines are one-half (the postsynaptic half) of most excitatory synapses. Ever since the direct observation over a decade ago that spines can continually change size and shape, spine dynamics has been of great research interest, especially as a mechanism for structural synaptic plasticity. In concert with this ongoing spine dynamics, the stability of the synapse is also needed to allow continued, reliable synaptic communication. Various cell-adhesion molecules help to structurally stabilize a synapse and its proteins. Here, we review the effects of disrupting N-cadherin, a prominent trans-synaptic adhesion molecule, on spine dynamics, as reported in Mysore et al. (2007). We highlight the novel method adopted therein to reliably detect even subtle changes in fast and slow spine dynamics. We summarize the structural, functional, and molecular consequences of acute N-cadherin disruption, and tie them in, in a working model, with longer-term effects on spines and synapses reported in the literature.

Activity-regulated N-cadherin endocytosis.

Enduring forms of synaptic plasticity are thought to require ongoing regulation of adhesion molecules, such as N-cadherin, at synaptic junctions. Little is known about the activity-regulated trafficking of adhesion molecules. Here we demonstrate that surface N-cadherin undergoes a surprisingly high basal rate of internalization. Upon activation of NMDA receptors (NMDAR), the rate of N-cadherin endocytosis is significantly reduced, resulting in an accumulation of N-cadherin in the plasma membrane. Beta-catenin, an N-cadherin binding partner, is a primary regulator of N-cadherin endocytosis. Following NMDAR stimulation, beta-catenin accumulates in spines and exhibits increased binding to N-cadherin. Overexpression of a mutant form of beta-catenin, Y654F, prevents the NMDAR-dependent regulation of N-cadherin internalization, resulting in stabilization of surface N-cadherin molecules. Furthermore, the stabilization of surface N-cadherin blocks NMDAR-dependent synaptic plasticity. These results indicate that NMDAR activity regulates N-cadherin endocytosis, providing a mechanistic link between structural plasticity and persistent changes in synaptic efficacy.

Effects of N-cadherin disruption on spine morphological dynamics.

Structural changes at synapses are thought to be a key mechanism for the encoding of memories in the brain. Recent studies have shown that changes in the dynamic behavior of dendritic spines accompany bidirectional changes in synaptic plasticity, and that the disruption of structural constraints at synapses may play a mechanistic role in spine plasticity. While the prolonged disruption of N-cadherin, a key synaptic adhesion molecule, has been shown to alter spine morphology, little is known about the short-term regulation of spine morphological dynamics by N-cadherin. With time-lapse, confocal imaging in cultured hippocampal neurons, we examined the progression of structural changes in spines following an acute treatment with AHAVD, a peptide known to interfere with the function of N-cadherin. We characterized fast and slow timescale spine dynamics (minutes and hours, respectively) in the same population of spines. We show that N-cadherin disruption leads to enhanced spine motility and reduced length, followed by spine loss. The structural effects are accompanied by a loss of functional connectivity. Further, we demonstrate that early structural changes induced by AHAVD treatment, namely enhanced motility and reduced length, are indicators for later spine fate, i.e., spines with the former changes are more likely to be subsequently lost. Our results thus reveal the short-term regulation of synaptic structure by N-cadherin and suggest that some forms of morphological dynamics may be potential readouts for subsequent, stimulus-induced rewiring in neuronal networks.

Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function.

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.