The clustered protocadherin endolysosomal trafficking motif mediates cytoplasmic association

Background Clustered protocadherins (Pcdhs) are a large family of neural cadherin-like proteins encoded by individual exons located within three gene clusters. Each exon codes an extracellular, transmembrane, and proximal cytoplasmic domain. These “variable” regions may be spliced to a constant cytoplasmic moiety encoded at the end of a cluster. Pcdh extracellular domains mediate homophilic cell-cell binding but their cytoplasmic domains cause intracellular retention and may negatively regulate Pcdh cell-cell binding. Pcdhs can be found at the cell surface in neurons and other cells but are also, unlike classical cadherins, prominently trafficked to the endolysosome system. It was previously found that a segment within the variable portion of the Pcdh-γA3 cytoplasmic domain (VCD) was shown to be necessary for endolysosomal trafficking. Results Here it is shown that this same VCD segment can mediate cytoplasmic association among Pcdhs from the different clusters. Internal deletions within this VCD region (termed here the VCD motif) that disrupt the association altered trafficking of Pcdh-γA3 in the endolysosomal system while deletions outside VCD motif did not affect trafficking. Conclusions The results show that Pcdhs associate cytoplasmically via a motif within the VCD and that this is critical for Pcdh trafficking. Given that truncation at the VCD motif alters endolysosomal trafficking of Pcdhs, the VCD interaction described here may provide new insights into the dynamic nature of Pcdh mediated cell-cell interactions

Perinatal Pb2+ exposure alters the expression of genes related to the neurodevelopmental GABA-shift in postnatal rats

Background: Lead (Pb2+) is an environmental neurotoxicant that disrupts neurodevelopment, communication, and organization through competition with Ca2+ signaling. How perinatal Pb2+ exposure affects Ca2+-related gene regulation remains unclear. However, Ca2+ activates the L-Type voltage sensitive calcium channel β-3 subunit (Ca-β3), which autoregulates neuronal excitability and plays a role in the GABA-shift from excitatory-toinhibitory neurotransmission. Method: A total of eight females (n = 4 Control and n = 4 Perinatal) and four males (n = 2 Control and n = 2 Perinatal) rats were used as breeders to serve as Dams and Sires. The Dam’s litters each ranged from N = 6– 10 pups per litter (M = 8, SD = 2), irrespective of Pb2+ treatment, with a majority of males over females. Since there were more males in each of the litters than females, to best assess and equally control for Pb2+− and litter-effects across all developmental time-points under study, female pups were excluded due to an insufficient sample size availability from the litter’s obtained. From the included pup litters, 24 experimentally naïve male Long Evans hooded rat pups (Control N = 12; Pb2+ N = 12) were used in the present study. Brains were extracted from rat prefrontal cortex (PFC) and hippocampus (HP) at postnatal day (PND) 2, 7, 14 and 22, were homogenized in 1 mL of TRIzol reagent per 100 mg of tissue using a glass-Teflon homogenizer. Postcentrifugation, RNA was extracted with chloroform and precipitated with isopropyl alcohol. RNA samples were then re-suspended in 100 μL of DEPC treated H2O. Next, 10 μg of total RNA was treated with RNase-free DNase (Qiagen) at 37 °C for 1 h and re-purified by a 3:1 phenol/chloroform extraction followed by an ethanol precipitation. From the purified RNA, 1 μg was used in the SYBR GreenER Two-Step qRT-PCR kit (Invitrogen) for first strand cDNA synthesis and the quantitative real-time PCR (qRT-PCR). The effects of perinatal Pb2+ exposure on genes related to early neuronal development and the GABA-shift were evaluated through the expression of: Ca-β3, GABAAR-β3, NKCC1, KCC2, and GAD 80, 86, 65, and 67 isoforms. Results: Perinatal Pb2+ exposure significantly altered the GABA-shift neurodevelopmental GOI expression as a function of Pb2+ exposure and age across postnatal development. Dramatic changes were observed with Ca-β3 expression consistent with a Pb2+ competition with L-type calcium channels. By PND 22, Ca-β3 mRNA was reduced by 1-fold and 1.5-fold in PFC and HP respectively, relative to controls. All HP GABA-β3 mRNA levels were particularly vulnerable to Pb2+ at PND 2 and 7, and both PFC and HP were negatively impacted by Pb2+ at PND 22. Additionally, Pb2+ altered both the PFC and HP immature GAD 80/86 mRNA expression particularly at PND 2, whereas mature GAD 65/67 were most significantly affected by Pb2+ at PND 22. Conclusions: Perinatal Pb2+ exposure disrupts the expression of mRNAs related to the GABA-shift, potentially altering the establishment, organization, and excitability of neural circuits across development. These findings offer new insights into the altered effects Pb2+ has on the GABAergic system preceding what is known regarding Pb2+ insults unto the glutamatergic system

Ubiquitination of the protocadherin-γA3 variable cytoplasmic domain modulates cell-cell interaction

The family of ∼60 clustered protocadherins (Pcdhs) are cell adhesion molecules encoded by a genomic locus that regulates expression of distinct combinations of isoforms in individual neurons resulting in what is thought to be a neural surface “barcode” which mediates same-cell interactions of dendrites, as well as interactions with other cells in the environment. Pcdh mediated same-cell dendrite interactions were shown to result in avoidance while interactions between different cells through Pcdhs, such as between neurons and astrocytes, appear to be stable. The cell biological mechanism of the consequences of Pcdh based adhesion is not well understood although various signaling pathways have been recently uncovered. A still unidentified cytoplasmic regulatory mechanism might contribute to a “switch” between avoidance and adhesion. We have proposed that endocytosis and intracellular trafficking could be part of such a switch. Here we use “stub” constructs consisting of the proximal cytoplasmic domain (lacking the constant carboxy-terminal domain spliced to all Pcdh-γs) of one Pcdh, Pcdh-γA3, to study trafficking. We found that the stub construct traffics primarily to Rab7 positive endosomes very similarly to the full length molecule and deletion of a substantial portion of the carboxy-terminus of the stub eliminates this trafficking. The intact stub was found to be ubiquitinated while the deletion was not and this ubiquitination was found to be at non-lysine sites. Further deletion mapping of the residues required for ubiquitination identified potential serine phosphorylation sites, conserved among Pcdh-γAs, that can reduce ubiquitination when pseudophosphorylated and increase surface expression. These results suggest Pcdh-γA ubiquitination can influence surface expression which may modulate adhesive activity during neural development

Puzzling It Out: The Current State of Scientific Knowledge on Pre-Kindergarten Effects - A Consensus Statement

Scientific research has established that if all children are to achieve their developmental potential, it is important to lay the foundation during the earliest years for lifelong health, learning, and positive behavior. A central question is how well our public pre-kindergarten (pre-K) programs are doing to build this foundation.Forty-two states and the District of Columbia, through 57 pre-K programs, have introduced substantial innovations in their early education systems by developing the infrastructure, program sites, and workforce required to accommodate pre-K education. These programs now serve nearly 30 percent of the nation's 4-year-olds and 5 percent of 3-year-olds

Hyperphosphorylation of Tau Associates With Changes in Its Function Beyond Microtubule Stability

Tau is a neuronal microtubule associated protein whose main biological functions are to promote microtubule self-assembly by tubulin and to stabilize those already formed. Tau also plays an important role as an axonal microtubule protein. Tau is an amazing protein that plays a key role in cognitive processes, however, deposits of abnormal forms of tau are associated with several neurodegenerative diseases, including Alzheimer disease (AD), the most prevalent, and Chronic Traumatic Encephalopathy (CTE) and Traumatic Brain Injury (TBI), the most recently associated to abnormal tau. Tau post-translational modifications (PTMs) are responsible for its gain of toxic function. Alonso et al. (1996) were the first to show that the pathological tau isolated from AD brains has prion-like properties and can transfer its toxic function to the normal molecule. Furthermore, we reported that the pathological changes are associated with tau phosphorylation at Ser199 and 262 and Thr212 and 231. This pathological version of tau induces subcellular mislocalization in cultured cells and neurons, and translocates into the nucleus or accumulated in the perinuclear region of cells. We have generated a transgenic mouse model that expresses pathological human tau (PH-Tau) in neurons at two different concentrations (4% and 14% of the total endogenous tau). In this model, PH-Tau causes cognitive decline by at least two different mechanisms: one that involves the cytoskeleton with axonal disruption (at high concentration), and another in which the apparent neuronal morphology is not grossly affected, but the synaptic terminals are altered (at lower concentration). We will discuss the putative involvement of tau in proteostasis under these conditions. Understanding tau’s biological activity on and off the microtubules will help shed light to the mechanism of neurodegeneration and of normal neuronal function

Characterization of MSB Synapses in Dissociated Hippocampal Culture with Simultaneous Pre- and Postsynaptic Live Microscopy

Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity

New Insights on the Sequence Stratigraphic Architecture of the Dakota Formation in Kansas–Nebraska–Iowa from a Decade of Sponsored Research Activity

The Cretaceous Dakota Formation in the areas of Kansas, Nebraska, and Iowa contains a rich and well-preserved microflora of fossil palynomorphs. A comprehensive listing of these taxa is presented in this publication as part of a continuing effort to develop a refined biostratigraphic scheme for mid-Cretaceous terrestrial deposits in North America. The Dakota Formation in this region contains four distinctive Albian-Cenomanian palynostratigraphic zones that are used to partition the unit into successive depositional cycles, and each zone records deposition in fluvial-estuarine environments. The late Albian Kiowa-Skull Creek depositional cycle at the base of the Dakota Formation is recognized throughout the study area, and is also recognized in other parts of the Cretaceous North American Western Interior basin. The overlying newly recognized latest Albian "Muddy-Mowry Cycle" is formally defined for the first time in this paper and correlates with depositional cycles recognized by other workers in other parts of the Western Interior basin. The Cenomanian lower Greenhorn Cycle is already widely recognized by many other workers throughout the Western Interior basin. Laterally extensive thin zones of pervasive carbonate mineral cementation are noted in fluvial-estuarine deposits in the Dakota Formation. They are believed to have formed as synsedimentary cements that precipitated below estuarine marine-flooding surfaces in settings related to discharging paleoground waters. The existence of these early diagenetic cementation zones has important implications for the recognition of diagenetic barriers and baffles to modern fluid flow in the Dakota Formation. New stable isotopic data on these authigenic cements are reported in this paper and add to a body of published data on the δ18O of mid-Cretaceous paleoprecipitation in North America