05 for events, p value = 0.01 for genes). Moreover, doubly transmitted events occur more often when the sib is female than male (p value = 0.09 for events, p value = 0.02 for genes). Recalling that the SSC cohort excluded families

with two affected children, these transmission biases are joint and independent evidence that there are fewer transmissions of ultrarare events to a male sib than to the autistic child in our cohort and support the hypothesis that a portion of ultrarare transmission Veliparib purchase events are causal in males. There appears to be no gender bias in the parent of origin of ultrarare events. Overall, the sources of transmissions of ultrarare events were 233 from the fathers and 223 from the mothers. For events that were transmitted but not to the unaffected male

siblings, the sources were 125 from the fathers and 125 from the mothers. The possible implications for this observation will be discussed later. We combined evidence from all CNVs, exploring transmitted events that overlap de novo events (Table S3). We also compiled lists of transmitted events with boundaries similar to those found in de novo events (Tables S5 and S10). Because ultrarare transmitted events and de selleck screening library novo events are sparse data sets, we cannot expect to draw strong conclusions for specific loci by combining these data. Rather, in these tables one can find anecdotal information that informally raises or lowers the suspicion that various loci are contributory. For Non-specific serine/threonine protein kinase example, transmission data raise the suspicion for USP7, CTNNA3, and genes encoding several related voltage-gated calcium channels (see next section) but diminish suspicion for the loci at NIPA (15q11.2) and NPHP1 (2q13). The latter loci appear to be mainly

unstable, and parental variants transmit equally to sibs and probands. Although our focus has been on rare variants that contribute to phenotypes in a dominant fashion, it has been documented that some autism can result from the combined action of recessive alleles (Morrow et al., 2008). Therefore, we scanned the genomes of probands and sibs looking for rare homozygous deletions that hit both alleles. Two were found, both occurring in probands (Figure S2). One disrupted COMMD1 (2p15) in a female. Homozygous loss of this gene is implicated in copper toxicosis in dogs ( van De Sluis et al., 2002) but has not been previously reported in humans. The deletion initially appeared as a de novo event; the father, but not the mother, carried a hemizygous deletion. However, the boundaries of the homozygous loss in the child matched those of the hemizygous loss in the father precisely, which raised our suspicion that the child had an instance of a rare but known occurrence of uniparental disomy of chromosome 2 ( Kotzot and Utermann, 2005).

The amplitude of the response depends upon the rates of

GPCR activation and deactivation and any inherent nonlinearities imposed by spatial compartmentalization or signal saturation (e.g., Ramanathan et al., 2005). In retinal rod photoreceptors, a single activated GPCR, rhodopsin (R∗), drives the signaling cascade that decreases cGMP and its associated inward cation current in a manner that is highly reproducible from trial to trial (e.g., Rieke and Baylor, 1998). The amplitude of the single-photon response (SPR) is considered a key factor in overcoming intrinsic cellular noise and thus for reliable transmission through the visual pathway (Field see more et al., 2005). There has been much progress in understanding the molecular basis of the amplification and deactivation steps that underlie the rod SPR (reviewed in Burns and Pugh, 2010). In the initial amplifying step, a R∗ activates G proteins at a rate of several hundred per second (Leskov et al., 2000; Heck and Hofmann, 2001) until R∗ is deactivated by phosphorylation by GRK1 and arrestin-1 binding (Kühn and Wilden, 1987). The second major amplifying step arises from cGMP Selleck Doxorubicin hydrolysis by the activated G protein-PDE6 enzyme complex (G∗-E∗), whose activity persists until deactivation by GTP hydrolysis catalyzed by

the RGS9 complex (He et al., 1998; Makino et al., 1999; Hu and Wensel, 2002). Although rapid R∗ and G∗-E∗ deactivation are required for normal recovery of the SPR, they are not sufficient; the fall in intracellular calcium that accompanies the SPR Resminostat must activate the synthesis of cGMP through guanylate cyclase activating proteins (GCAPs). Abolishing calcium feedback via GCAPs increases the amplitude and slows the recovery of the SPR (Mendez et al., 2001; Burns et al., 2002). In addition, loss of feedback via GCAPs increases the intrinsic cellular noise in a manner that can impair transmission at the rod-to-rod bipolar synapse and behavioral performance at visual threshold (Okawa et al., 2010). Genetic perturbations of R∗

and G∗-E∗ deactivation also produce dramatic changes in the overall time course of the rod photoresponse. Nonetheless, the SPRs of rods with defective deactivation have average peak amplitudes very close to those of wild-type rods. For example, preventing rhodopsin phosphorylation slows the rate of R∗ deactivation 75-fold, from a normal average lifetime of ∼40 ms (Gross and Burns, 2010) to about 3 s (Grk1−/−; Chen et al., 1999), yet the amplitude of the SPR is increased by a factor of only two. Similarly, abolishing expression of the RGS9 complex slows G∗-E∗ deactivation about 10-fold yet has only a subtle effect on the SPR amplitude ( Chen et al., 2000; Krispel et al., 2003; Keresztes et al., 2004).

The potential benefits of muscle stretching for cramp prevention remain unknown to large numbers of patients (Blyton et al 2012), suggesting that wider recognition of the usefulness of prophylactic stretching may well improve the quality of life for many patients. “
“Thirty-four years ago Australian Journal of Physiotherapy published an article by Prue Galley, FG-4592 research buy a dynamic and passionate physiotherapist, entitled ‘Patient referral and the physiotherapist’ ( Galley 1976). This article was a synthesis of the debates and arguments that were raging at the time about whether Australian physiotherapists were ready to act as primary contact professionals. Galley asked: Have we

as physiotherapists, the knowledge, the courage, the will and the vision, to take this independent Crenolanib clinical trial step, knowing full well that it will involve increased responsibility, greater dedication, and selfdiscipline from us all? The profession responded in the affirmative and on 14 August 1976 the Australian Physiotherapy Association repealed our first ethical principle which stated that ‘It is unethical for a member to act in a professional capacity except on referral by a registered medical or dental practitioner’. The move to become primary

contact professionals was perhaps the most significant move in the over hundred year history of the profession. This was a change not taken lightly but one that grew out of a sense that the profession had matured and that it was time to move beyond our close association with the medical profession. At the time this action by Australia caused significant argument in the world physiotherapy community as we were the first country to enact this change. Not all countries were comfortable with the move as a subordinate role to the medical profession was the preferred model for physiotherapy practice in some countries. The matter was scheduled for discussion at the World Congress of Physical Therapy (WCPT) 8th General Congress held in Tel Aviv. The

Australian Histone demethylase delegation went to Israel in 1978 with a proposal designed to enable each member country to set its own standards in this regard. Australia expected to encounter significant resistance – to the point that the Association was prepared to be expelled from WCPT if the motion did not pass. Fortunately that did not occur, and through sustained lobbying and advocacy the delegates succeeded in their mission. The meeting passed the Australian resolution that ‘the issue of primary practitioner status be interpreted by each country in terms of their own standards’. In 1995 this belief was strengthened by the WCPT Declaration of Principle on Autonomy which states ‘Patients/clients should have direct access to physical therapist services’. Three decades later primary contact status has moved from being an issue which nearly split the international community apart to one which is bringing the disparate WCPT member associations together.

2 μg/mL, CNIH-2; 1:50, pan-Type I TARP) in D-PBS plus 2% normal goat serum. Cultures were rinsed and incubated with fluorescence-conjugated secondary antibodies (Invitrogen, 1:500) in D-PBS for 1 hr at room temperature. After a final rinse, coverslips were mounted and imaged using Leica immunofluorescence

microscope systems (Wetzlar, Germany). Rat hippocampal slices (400 μm) were incubated in slicing buffer (in mM: 124 NaCl, 26 mM NaHCO3, 3 KCl, 10 Glucose, 0.5 CaCl2, and 4 MgCl2) for 1 hr. Slices were then placed into biotinylation solution (biotinylation solution = slicing solution except [CaCl2] and [MgCl2] were raised to 2.3 and 1.3 mM, respectively) ∼4°C biotinylation solution for 5 min. Surface proteins of the dissected

were labeled with sulfo NHS ATM/ATR cancer Sirolimus purchase SS biotin (1.5 mg/mL; Pierce) for 30 min on ice and the reaction quenched with glycine (50 mM). Hippocampi were homogenized with Tris buffer (TB: 50 mM Tris, pH 7.4, 2 mM EGTA) then sonicated. Homogenates were centrifuged at 100,000 × g for 20 min and the pellet was resuspended in TB containing NaCl (TN: TB + 100 mM NaCl). 50% ULTRA link Neutravidin (Roche) was added and incubated at 4°C for 2 hr. Nonbound internal protein solution was removed. Beads were washed with RIPA buffer and biotinylated surface proteins were eluted by boiling for 5 min in Laemmli buffer containing DTT (7.7 mg/mL). Eluted proteins and internal proteins were separated by SDS-PAGE and detected via western blotting. Data are represented as mean ± SEM and are the result of at least three independent experiments. Analyses involving three or more data sets were performed with a one-way ANOVA with a Tukey-Kramer post-hoc

analysis using GraphPad Prism software (Carlsbad, CA). Analyses involving two data sets were performed with an uncorrected Student’s t test or with a Student’s t test with a Welsh correction, only if the variances were statistically different. Significance was set as a p-value of less than 0.05. A.S.K., M.B.G., H.Y., Y.T., E.R.S., H.W., Y.-W.Q., E.S.N., and D.S.B. are full-time Idoxuridine employees of Eli Lilly and Company. This work was supported in part by grants to S.T. from the NIMH (R01MH077939) and the NINDS (RC1NS068966). “
“Neuronal somata and dendrites acidify when depolarized by trains of action potentials and voltage-clamp pulses (Ahmed and Connor, 1980, Trapp et al., 1996a, Trapp et al., 1996b and Willoughby and Schwiening, 2002), elevated extracellular [K+] (OuYang et al., 1995, Zhan et al., 1998 and Yu et al., 2003), or glutamate agonists (Vale-González et al., 2006 and Bolshakov et al., 2008). Most studies suggest that this depolarization-induced cytosolic acidification results from Ca2+ influx-mediated activation of the plasmalemmal Ca2+ ATPase, which imports H+ as it extrudes Ca2+ (Schwiening and Thomas, 1998 and Chesler, 2003).

A key issue, therefore, is whether the NMDAR content is altered at individual synapses. We first addressed this functionally, by collecting mixed spontaneous AMPAR- and NMDAR-mediated High Content Screening currents at −70 mV in the absence of external Mg2+, then washing on APV and collecting the pure AMPAR-mediated currents. The pure AMPAR currents were then subtracted from the mixed currents to give a pure NMDAR-mediated spontaneous current. We performed these experiments using simultaneously recorded NLGN1 miR-expressing neurons and neighboring control cells in the dentate gyrus and collected both evoked and spontaneous currents, using the evoked currents to assess the validity

of the technique. The stimulation-evoked, subtracted NMDAR-mediated currents in NLGN1 miR expressing cells were reduced, as expected, compared to control cells (Figures 2A and 2B).

Moreover, the magnitude of the reduction was identical to that found when NMDAR currents were measured at +40 mV in the previous experiment (as percent selleck chemicals llc of control, +40 mV, 32.12 ± 5.26; subtracted 23.4 ± 4.92; p > 0.05), thus providing validation of the technique. Furthermore, neither the charge transfer of the NMDAR current as a percent of the total charge transfer of the mixed AMPAR/NMDAR current nor the kinetics of the NMDAR current were altered in the evoked Amisulpride response (Figures 2C and 2D). We next analyzed the spontaneous currents in these same cells (Figure 2E) and found a dramatic reduction in the frequency of spontaneous events (Figure 2F), but no

change in amplitude of either the mixed current, the pure AMPAR current, or the pure, subtracted NMDAR current (Figure 2G). Like the evoked current, knockdown did not affect the percentage of spontaneous charge transfer accounted for by NMDA current (Figure 2H). We consequently conclude that the reduction in evoked NMDAR currents is functionally due to an all-or-none loss of synapses, while the remaining synapses have normal numbers of NMDARs. To complement the functional evidence for an all-or-none loss of synapses following neuroligin knockdown, we examined spine density. Following knockdown of NLGN1, we filled transduced dentate granule cells and neighboring control cells with fluorescent dye and imaged their dendrites (Figure 2I). We observed a reduction in spine density in NLGN1 miR expressing cells as compared to control (Figure 2J) of a similar magnitude to the reduction in evoked currents. Spine density in dentate granule cells following the knockdown of NLGN3 was also reduced, confirming that synaptic loss is a general response to neuroligin knockdown (Figures S2A and S2B). Finally, we performed a coefficient of variation analysis on the paired evoked recordings following neuroligin knockdown.

Furthermore, the photoreceptor cells displayed extensive ER membrane accumulations and dilated Golgi ( Figure 7A), consistent with aggregation of TRP and Rh1 in the secretory pathway. At 2 weeks, the xport1 mutant photoreceptor cells were severely degenerated. The rhabdomeres of all eight

photoreceptors were vastly reduced and many were completely missing ( Figures 6C and 6D). To assess whether the retinal degeneration was enhanced by light stimulation of phototransduction, we reared the xport1 mutant for 2 weeks in constant darkness. Dark-reared flies still showed ER membrane accumulations and dilated Golgi ( Figure 7D), but now exhibited nearly normal rhabdomere morphology ( Figure 6E). Therefore activation of phototransduction by light enhances the retinal degeneration in xport1 buy Screening Library mutants. The retinal pathology was fully rescued by the expression of wild-type XPORT in the xport1 mutant ( Figure 6F). The molecular mechanisms underlying retinal degeneration are diverse and have been well studied in the Drosophila visual system. Two well-characterized mechanisms involve Capmatinib either (1) accumulation of Rh1 in the secretory pathway due to defective folding/trafficking or (2) unregulated Ca2+ levels due to defective phototransduction ( Colley, 2010, Rosenbaum et al., 2006 and Wang and Montell, 2007). The finding that

light significantly enhanced the retinal degeneration in the xport1 mutant is contrasted to other known mutants defective in Rh1 maturation,

for which the retinal degeneration is light-independent ( Colley et al., 1991, Colley et al., 1995, Kurada and O’Tousa, 1995 and Webel Metalloexopeptidase et al., 2000). However, the xport1 mutant is unique in that it displays defects in both protein trafficking and TRP channel function. Loss of TRP channel expression can lead to a retinal degeneration unrelated to protein trafficking ( Wang and Montell, 2007). In this instance, the retinal degeneration is light-dependent and is triggered by defects in calcium influx through the light-sensitive TRP channels. Given that the retinal degeneration in xport1 is light-enhanced, we investigated the relative contribution of protein trafficking defects versus the lack of TRP channel function to the overall retinal degeneration. To accomplish this, we took advantage of two retinal degeneration mutants, ninaE318 and trp343. The ninaE318 mutant exhibited a severe reduction in Rh1 and displayed defects in Rh1 transport through the secretory pathway ( Figures S5A and S5C). However, TRP protein levels were wild-type in ninaE318 ( Figure S5B). Therefore, ninaE318 exhibits a retinal degeneration that is due solely to defects in protein trafficking. In contrast, the trp343 mutant was null for TRP protein ( Figure S5B) but Rh1 levels were wild-type and Rh1 specifically localized to the rhabdomeres ( Figures S5A and S5C).

This raises the question as to how these signaling pathways interact at DMH synapses. If CB1R activation at the presynaptic terminal precludes the effects of NO to enhance GABA release, then the application of a CB1R agonist should block the potentiation of GABA transmission by the NO donor, SNAP. Consistent with this

idea, SNAP failed to increase evoked IPSC amplitude when applied to slices that were continuously perfused with WIN 55,212-2 (104% ± 12.6% of WIN 55,212-2, n = 5, p = 0.646; Figures 5A and 5C). Similarly, it did not affect PPR (baseline: 0.961 ± 0.119; post-drug: 0.883 ± CAL-101 concentration 0.178; p = 0.544) or CV (baseline: 0.502 ± 0.071; post-drug: 0.500 ± 0.045; p = 0.962). Conversely, WIN 55,212-2 still effectively depressed IPSCs that were first potentiated by SNAP (36% ± 12.0% of SNAP,

n = 7; Figures 5B and 5D). This change was accompanied by an increase in PPR (baseline: 0.663 ± 0.109; post-drug: 0.950 ± 0.099; p = 0.048) and CV (baseline: 0.332 ± 0.084; post-drug: 0.593 ± 0.117; p = 0.049), consistent with the effect of WIN 55,212-2 in the absence of SNAP. Interestingly, the onset of the WIN 55,212-2–induced PD0325901 purchase depression was accelerated in the presence of SNAP when compared with WIN 55,212-2 alone, as evidenced by a decrease in the decay constant of the depression after drug application by approximately 80% (from 13.0 ± 2.8 min to 2.5 ± 0.7 min; Figure 5D). These data suggest that activation of CB1Rs attenuates the NO-induced increase in GABA release, whereas NO itself enhances the effects of a CB1R ligand. Next, we conducted experiments to determine the consequences of NO production on eCB-mediated LTDGABA. When NO synthesis was inhibited by L-NAME, HFS (100 Hz for 4 s ×

2, 0.05 Hz interval) failed to elicit LTDGABA (111% ± 11.3% of baseline, n = 8, p = 0.350; Figure 5E), the changes in PPR (baseline: 0.860 ± 0.086; post-HFS: 0.826 ± 0.102; p = 0.369), or the changes in CV (baseline: 0.311 ± 0.028; post-HFS: 0.336 ± first 0.07; p = 0.452). This suggests that NO signaling is required either for eCB production or CB1R signaling. Consistent with the latter idea, direct activation of CB1Rs by WIN 55,212-2 in the presence of L-NAME failed to significantly depress evoked IPSC amplitude (88% ± 10.8% of baseline, n = 6, p = 0.375; Figure 5F), PPR (baseline: 0.903 ± 0.129; post-drug: 0.889 ± 0.092; p = 0.850), or CV (baseline: 0.362 ± 0.067; post-drug: 0.410 ± 0.094; p = 0.168). Overall, these data point to an inherent complexity in the signaling of the retrograde transmitters eCBs and NO in the DMH. Specifically, they argue that eCB signaling prevents NO-mediated potentiation of GABA synapses but that NO signaling is required for eCB-induced depression of GABA signaling. We have demonstrated thus far that CB1R signaling precludes NO-mediated LTPGABA in the DMH. Here, we hypothesized that a physiological state in which CB1R signaling is compromised should favor the induction of LTPGABA.

Any transition in muscular Selisistat work from rest to short duration bouts of movement requires a rapid adjustment in ATP synthesis in order to match ATP usage.33 This is met initially by the ATP-CP system and anaerobic glycolysis. If the activity remains low intensity, reliance on anaerobic glycolysis is fleeting and oxidative metabolism attends to ATP synthesis. If PA is performed at higher intensities, the contribution of anaerobic glycolysis becomes more significant.

Pulmonary oxygen uptake (VO2) has traditionally been used as a marker of oxidative metabolism and it is generally assumed that VO2 is linearly related to work rate, and at a given intensity, VO2 remains constant.34 These assumptions are however, not strictly true. The pulmonary oxygen uptake kinetic response to exercise has fast (primary) Nutlin-3 cell line and slow components, reflecting the efficiency of skeletal muscle oxidative metabolism, the relative degree of fatigue and affording an understanding of the interplay between cardiopulmonary and metabolic processes during PA and how these may be affected by conditions such as obesity.35 The pulmonary

oxygen uptake kinetic response to low-to-moderate intensity exercise (i.e., intensity below the gas exchange threshold) has been described by three phases. Phase I begins as soon as the child transits from a period of rest to a bout of PA. First there is a delay, followed by a rapid rise in oxygen uptake. This first phase is also known as the cardiodynamic phase and reflects cardiovascular and pulmonary adaptations. Phase I is not dependent on VO2, rather it is largely a marker of the increase in pulmonary blood flow. Phase I is followed by an exponential increase in VO2 (phase II) that drives VO2 to steady state (phase III). Phase II kinetics are also known as the primary (fast) component and are described by a time constant revealing the time taken to achieve 63% of the change in oxygen uptake. Thiamine-diphosphate kinase The primary component provides a very close reflection (within about 10%) of the kinetics of oxygen uptake at the muscle. In phases I and

II, when ATP re-synthesis cannot be fully supported by oxidative phosphorylation, the additional energy requirements are met from oxygen stores, PCr, and glycolysis. The oxygen equivalent of these energy sources is known as the oxygen deficit and the faster the time constant the smaller the oxygen deficit.35 During higher intensity PA, activity that is above the gas exchange threshold, a slow component manifests at phase III. During low to moderate intensity activity, there is no phase III slow component, with VO2 attaining steady-state at the end of phase II. In comparison, during more vigorous intensity activity above the gas exchange threshold VO2 continues to rise above what would have been steady-state and this reflects a loss of muscle efficiency and ensuing fatigue.

However, knowledge of the existence of these relatively low-dimensional patterns of activity provides a general way to understand how information propagates from the AL to their

followers, the KCs of the MB. KCs are sensitive to coincidence in presynaptic input (Perez-Orive et al., 2002 and Perez-Orive et al., 2004). If KCs receive identical synchronized input from PNs during every cycle of the oscillation, the same set of KCs will be activated repeatedly over the duration of the odor presentation. However, experimental recordings show that KCs generate very few spikes (∼2–3) during the odor presentation. The absence DAPT of LN-LN interactions would therefore compromise the temporal sparseness of the odor representation by KCs. A number of algorithms to color random graphs exist. However, except under special circumstances, these algorithms do not guarantee that the coloring will always be minimal or that all possible colorings of the network will be obtained in a reasonable length of time (Kubale, 2004). selleck inhibitor Given the complexity of the graph coloring problem, using random graphs as our starting point would have been impractical. Hence we chose to construct

graphs in which neurons associated with a particular color were connected to all neurons associated with other colors. How well do these constructed networks emulate many the dynamics of realistic random networks? In the networks constructed thus far, each neuron received an equal number of connections as all other neurons that were affiliated with the same color. In realistic random networks this assumption is not true in general. Variability in input across LNs can cause the dynamics of the network to deviate from the dynamics predicted by the networks we simulated. To test the effect of

perturbations to the network structure, we simulated a network consisting of two groups of fifteen neurons that were reciprocally connected to each other (Figure 6A). Neurons in each group extended 1–14 connections to neurons belonging to the other group. This is the widest possible variability in connections that can be achieved in this network while ensuring that no neuron is isolated from the network. In addition a network constructed in this manner is also guaranteed to possess a chromatic number two. First, we reordered the rows and columns of the adjacency matrix of the network such that neurons affiliated with the same color were grouped together (Figure 6B). As in previous examples, the adjacency matrix of the random network consisted of diagonal blocks of zeros. However, all elements of the off-diagonal blocks are not uniformly one.

47 ± 0.11; MMP9/MMP13i, 0.27 ± 0.11; MMP13i, 0.92 ± 0.15; Figures S3C and S3D), indicating that MMP9-dependent cleavage is active not only during periods of elevated neuronal activity, but also under basal conditions. Moreover, we noted that MMP3 inhibitor III (50 μM) induced a partial but nonsignificant decrease in NLG1-NTFs after KCl (KCl + MMP3i, 1.7 ± 0.1, p = 0.092; Figures 3E and 3F). MMPs are secreted as inactive zymogens and require

cleavage of their prodomains to become enzymatically active (Ethell and Ethell, 2007). Thus, the effect of MMP3 inhibition could be due to impaired MMP9 activation. To determine which MMP is the terminal protease-cleaving NLG1, we treated neurons with 4-aminophenylmercuric acetate (APMA), a check details compound that nonselectively activates all MMPs by cleaving their prodomains (Van Wart and Birkedal-Hansen, selleck compound 1990) and tested the effect of specific MMP inhibitors. Brief incubation of DIV21 cortical neurons with 0.5 mM APMA for 15 min induced robust generation of NLG1-NTFs (3.8 ± 0.8-fold increase relative to control; Figures 3G and 3H). Coincubation with MMP2/MMP9 inhibitor II (0.3 μM) blocked APMA-induced cleavage (0.6 ± 0.1), whereas MMP2 inhibitor III (50 μM) and MMP3 inhibitor III (50 μM) had no effect (3.8 ± 1.0 and 3.2 ± 0.8, respectively),

indicating that MMP9 is the downstream protease responsible for cleavage of NLG1. To further validate these findings, we tested how NLG1 is regulated by activity in neurons lacking MMP9. KCl depolarization of DIV17 and DIV18 wild-type (WT) mouse cortical cultures for 2 hr resulted in extensive loss of NLG1 (0.36 ± 0.01 relative to control; Figure 3I). By contrast, KCl incubation of MMP9 KO cultures induced no loss of NLG1 (0.90 ± 0.04 relative to control; Figure 3J), confirming that MMP9 is responsible for activity-dependent regulation of NLG1. To characterize the activity-dependent production of NLG1-CTFs, we measured CTF levels in whole cell extracts of DIV21 dissociated

neuron cultures treated with KCl. As expected, KCl incubation increased NLG1-CTF levels (1.6 ± 0.4 compared to control; Figures S3E and S3F). Interestingly, inhibition of the γ-secretase complex with DAPT (20 μM) during KCl treatment resulted in increased accumulation of NLG1-CTFs (KCl+DAPT, 3.7 ± 0.8; Figures S3E and S3F), indicating that NLG1 is processed by the γ-secretase complex following ectodomain cleavage. Deglycosylation of NLG1-NTFs nearly produces ∼70 kDa species (Figure 2E), which, based on amino acid mass, indicates that proteolysis occurs in the extracellular juxtamembrane region of NLG1. To determine the specific domain targeted for cleavage, we generated a series of mutants with sequential deletions and amino acid (aa) replacements in the juxtamembrane domain (Figure 4A). NLG1 mutants were screened for their resistance to APMA cleavage using biotinylation-based labeling and isolation of NLG1-NTFs in COS7 cells. Brief incubation with APMA resulted in robust shedding of GFP-NLG1 (Figure 4B).