Secretagogin marks amygdaloid PKCδ interneurons and modulates NMDA receptor availability.

The perception of and response to danger is critical for an individual's survival and is encoded by subcortical neurocircuits. The amygdaloid complex is the primary neuronal site that initiates bodily reactions upon external threat with local-circuit interneurons scaling output to effector pathways. Here, we categorize central amygdala neurons that express secretagogin (Scgn), a Ca2+-sensor protein, as a subset of protein kinase Cδ (PKCδ)+ interneurons, likely "off cells." Chemogenetic inactivation of Scgn+/PKCδ+ cells augmented conditioned response to perceived danger in vivo. While Ca2+-sensor proteins are typically implicated in shaping neurotransmitter release presynaptically, Scgn instead localized to postsynaptic compartments. Characterizing its role in the postsynapse, we found that Scgn regulates the cell-surface availability of NMDA receptor 2B subunits (GluN2B) with its genetic deletion leading to reduced cell membrane delivery of GluN2B, at least in vitro. Conclusively, we describe a select cell population, which gates danger avoidance behavior with secretagogin being both a selective marker and regulatory protein in their excitatory postsynaptic machinery.

Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

Conventional measures of intrinsic excitability are poor estimators of neuronal activity under realistic synaptic inputs.

Activity-dependent regulation of intrinsic excitability has been shown to greatly contribute to the overall plasticity of neuronal circuits. Such neuroadaptations are commonly investigated in patch clamp experiments using current step stimulation and the resulting input-output functions are analyzed to quantify alterations in intrinsic excitability. However, it is rarely addressed, how such changes translate to the function of neurons when they operate under natural synaptic inputs. Still, it is reasonable to expect that a strong correlation and near proportional relationship exist between static firing responses and those evoked by synaptic drive. We challenge this view by performing a high-yield electrophysiological analysis of cultured mouse hippocampal neurons using both standard protocols and simulated synaptic inputs via dynamic clamp. We find that under these conditions the neurons exhibit vastly different firing responses with surprisingly weak correlation between static and dynamic firing intensities. These contrasting responses are regulated by two intrinsic K-currents mediated by Kv1 and Kir channels, respectively. Pharmacological manipulation of the K-currents produces differential regulation of the firing output of neurons. Static firing responses are greatly increased in stuttering type neurons under blocking their Kv1 channels, while the synaptic responses of the same neurons are less affected. Pharmacological blocking of Kir-channels in delayed firing type neurons, on the other hand, exhibit the opposite effects. Our subsequent computational model simulations confirm the findings in the electrophysiological experiments and also show that adaptive changes in the kinetic properties of such currents can even produce paradoxical regulation of the firing output.

Protein kinase D exerts neuroprotective functions during oxidative stress via nuclear factor kappa B-independent signaling pathways.

Members of the protein kinase D (PKD) family of serine/threonine kinases are known to exert diverse roles in neuronal stress responses. Here, we show the transient activation and nuclear translocation of endogenous PKD upon oxidative stress induced by H2 O2 treatment in primary neuronal cultures. Using pharmacological inhibition, we show that PKD activity protects neurons from oxidative stress-induced cell death. Although members of the canonical nuclear factor kappa-light-chain-enhancer of activated B cells (NF kappaB) pathway were phosphorylated upon H2 O2 treatment, it was found that the neuronal response to oxidative stress is not executed through the nuclear translocation and activity of RelA. On the other hand, we demonstrate for the first time in neuronal cells, the association of green fluorescent protein-tagged kinase inactive PKD1 with mitochondrial membranes in vivo and the presence of PKD activity in the close vicinity of mitochondria in vitro. Our findings thus support the notion that the neuroprotective role of PKD is exerted independently from NF kappaB signaling and suggest a potential mitochondrial function for PKD in cultured neurons.

Frequency-dependent regulation of intrinsic excitability by voltage-activated membrane conductances, computational modeling and dynamic clamp.

As one of the most unique properties of nerve cells, their intrinsic excitability allows them to transform synaptic inputs into action potentials. This process reflects a complex interplay between the synaptic inputs and the voltage-dependent membrane currents of the postsynaptic neuron. While neurons in natural conditions mostly fire under the action of intense synaptic bombardment and receive fluctuating patterns of excitation and inhibition, conventional techniques to characterize intrinsic excitability mainly utilize static means of stimulation. Recently, we have shown that voltage-gated membrane currents regulate the firing responses under current step stimulation and under physiologically more realistic inputs in a differential manner. At the same time, a multitude of neuron types have been shown to exhibit some form of subthreshold resonance that potentially allows them to respond to synaptic inputs in a frequency-selective manner. In this study, we performed virtual experiments in computational models of neurons to examine how specific voltage-gated currents regulate their excitability under simulated frequency-modulated synaptic inputs. The model simulations and subsequent dynamic clamp experiments on mouse hippocampal pyramidal neurons revealed that the impact of voltage-gated currents in regulating the firing output is strongly frequency-dependent and mostly affecting the synaptic integration at theta frequencies. Notably, robust frequency-dependent regulation of intrinsic excitability was observed even when conventional analysis of membrane impedance suggested no such tendency. Consequently, plastic or homeostatic regulation of intrinsic membrane properties can tune the frequency selectivity of neuron populations in a way that is not readily expected from subthreshold impedance measurements.

A new tool for the quantitative analysis of dendritic filopodial motility.

Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of mass (CoM) within a selected region based on the weighted intensity values of structure forming pixels, selected by upper and lower intensity thresholds. Using synthetic and real biological samples, we prove that the displacement of the weighted CoM reliably characterizes the motility of dendritic protrusions. Additionally, guidelines to define optimal parameters of live cell recordings from dendritic protrusions are provided. © 2014 International Society for Advancement of Cytometry.

Dendritic effects of genetically encoded actin-labeling probes in cultured hippocampal neurons.

Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labeled by genetically fusing fluorescent proteins to actin monomers, actin-binding proteins, or single-chain anti-actin antibodies. In the present study, we compared the dendritic effect of EGFP-actin, LifeAct-TagGFP2 (LifeAct-GFP), and Actin-Chromobody-TagGFP2 (AC-GFP) in mouse cultured hippocampal neurons using unbiased quantitative methods. The actin-binding probes LifeAct-GFP and AC-GFP showed similar affinity to F-actin, but in contrast to EGFP-actin, they did not reveal subtle changes in actin remodeling between mushroom-shaped spines and filopodia. All tested actin probes colocalized with phalloidin similarly; however, the enrichment of LifeAct-GFP in dendritic spines was remarkably lower compared with the other constructs. LifeAct-GFP expression was tolerated at a higher expression level compared with EGFP-actin and AC-GFP with only subtle differences identified in dendritic spine morphology and protrusion density. While EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP-expressing neurons displayed a reduced dendritic tree. Thus, although all tested actin probes may be suitable for actin imaging studies, certain limitations should be considered before performing experiments with a particular actin-labeling probe in primary neurons.

Endocytosis of AMPA receptors: Role in neurological conditions.

AMPA receptors are glutamate-gated ion channels, present in a wide range of neuron types and in glial cells. Their main role is to mediate fast excitatory synaptic transmission, and therefore, they are critical for normal brain function. In neurons, AMPA receptors undergo constitutive and activity-dependent trafficking between the synaptic, extrasynaptic and intracellular pools. The kinetics of AMPA receptor trafficking is crucial for the precise functioning of both individual neurons and neural networks involved in information processing and learning. Many of the neurological diseases evoked by neurodevelopmental and neurodegenerative malfunctions or traumatic injuries are caused by impaired synaptic function in the central nervous system. For example, attention-deficit/hyperactivity disorder (ADHD), Alzheimer's disease (AD), tumors, seizures, ischemic strokes, and traumatic brain injury are all characterized by impaired glutamate homeostasis and associated neuronal death, typically caused by excitotoxicity. Given the important role of AMPA receptors in neuronal function, it is not surprising that perturbations in AMPA receptor trafficking are associated with these neurological disorders. In this book chapter, we will first introduce the structure, physiology and synthesis of AMPA receptors, followed by an in-depth description of the molecular mechanisms that control AMPA receptor endocytosis and surface levels under basal conditions or synaptic plasticity. Finally, we will discuss how impairments in AMPA receptor trafficking, particularly endocytosis, contribute to the pathophysiology of various neurological disorders and what efforts are being made to therapeutically target this process.

LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization.

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.

Homeostatic plasticity and burst activity are mediated by hyperpolarization-activated cation currents and T-type calcium channels in neuronal cultures.

Homeostatic plasticity stabilizes neuronal networks by adjusting the responsiveness of neurons according to their global activity and the intensity of the synaptic inputs. We investigated the homeostatic regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) and T-type calcium (CaV3) channels in dissociated and organotypic slice cultures. After 48 h blocking of neuronal activity by tetrodotoxin (TTX), our patch-clamp experiments revealed an increase in the depolarizing voltage sag and post-inhibitory rebound mediated by HCN and CaV3 channels, respectively. All HCN subunits (HCN1 to 4) and T-type Ca-channel subunits (CaV3.1, 3.2 and 3.3) were expressed in both control and activity-deprived hippocampal cultures. Elevated expression levels of CaV3.1 mRNA and a selective increase in the expression of TRIP8b exon 4 isoforms, known to regulate HCN channel localization, were also detected in TTX-treated cultured hippocampal neurons. Immunohistochemical staining in TTX-treated organotypic slices verified a more proximal translocation of HCN1 channels in CA1 pyramidal neurons. Computational modeling also implied that HCN and T-type calcium channels have important role in the regulation of synchronized bursting evoked by previous activity-deprivation. Thus, our findings indicate that HCN and T-type Ca-channels contribute to the homeostatic regulation of excitability and integrative properties of hippocampal neurons.

The Plasma Membrane Ca2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton.

We demonstrated that the plasma membrane Ca2+ ATPase PMCA4b inhibits migration and metastatic activity of BRAF mutant melanoma cells. Actin dynamics are essential for cells to move, invade and metastasize, therefore, we hypothesized that PMCA4b affected cell migration through remodeling of the actin cytoskeleton. We found that expression of PMCA4b in A375 BRAF mutant melanoma cells induced a profound change in cell shape, cell culture morphology, and displayed a polarized migratory character. Along with these changes the cells became more rounded with increased cell-cell connections, lamellipodia and stress fiber formation. Silencing PMCA4b in MCF-7 breast cancer cells had a similar effect, resulting in a dramatic loss of stress fibers. In addition, the PMCA4b expressing A375 cells maintained front-to-rear Ca2+ concentration gradient with the actin severing protein cofilin localizing to the lamellipodia, and preserved the integrity of the actin cytoskeleton from a destructive Ca2+ overload. We showed that both PMCA4b activity and trafficking were essential for the observed morphology and motility changes. In conclusion, our data suggest that PMCA4b plays a critical role in adopting front-to-rear polarity in a normally spindle-shaped cell type through F-actin rearrangement resulting in a less aggressive melanoma cell phenotype.

Anoxia leads to a rapid translocation of human trypsinogen 4 to the plasma membrane of cultured astrocytes.

Trypsinogen 4 is specifically expressed in the human brain, mainly by astroglial cells. Although its exact role in the nervous tissue is yet unclear, trypsin 4-mediated pathological processes were suggested in Alzheimer's disease, multiple sclerosis and ischemic injury. In the present study, we analyzed the intracellular distribution of fluorescently tagged human trypsinogen 4 isoforms during normal and anoxic conditions in transfected mouse primary astrocytes. Our results show that initiation of anoxic milieu by the combined action of KCN treatment and glucose deprivation rapidly leads to the association of leader peptide containing trypsinogen 4 constructs to the plasma membrane. Using rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide), a synthetic chromogen peptide substrate of trypsin, we show that anoxia can promote extracellular activation of trypsinogen 4 indicating that extracellular activation of human trypsinogen 4 can be an important component in neuropathological changes of the injured human brain.

Short-term neuronal effects of fumonisin B1 on neuronal activity in rodents.

Fumonisin B1 (FB1) is a mycotoxin produced by microscopic fungi (mostly Fusarium species), which may infect our major crops. The toxin inhibits the development of these plants and may also have harmful effects on animals and humans consuming the infected crops. FB1 inhibits sphingolipid biosynthesis which leads to altered membrane characteristics and consequently, altered cellular functions. There are some indications that the toxin has inhibitory effects on neuronal activity in case of repeated consumption, presumably due to sphingolipid depletion. However, according to new literature data, FB1 may have acute excitatory neural effects, too, via different mechanisms of action. Therefore, in the present study, we addressed the neuronal network effects of FB1 following acute treatment, using different electrophysiological techniques in vitro and in vivo. Acute treatments with FB1 (10-100 µM) were carried out on brain slices, tissue cultures and live animals. After direct treatment of samples, electrically evoked or spontaneous field potentials were examined in the hippocampus and the neocortex of rat brain slices and in hippocampal cell cultures. In the hippocampus, a short-term increase in the excitability of neuronal networks and individual cells was observed in response to FB1 treatment. In some cases, the initially enhanced excitation was reversed presumably due to overactivation of neuronal networks. Normal spontaneous activity was found to be stimulated in hippocampal cell cultures. Seizure susceptibility was not affected in the neocortex of brain slices. For the verification of the results caused by direct treatment, effects of systemic administration of FB1 (7.5 mg/kg, i.p.) were also examined. Evoked field potentials recorded in vivo from the somatosensory cortex and cell activation measured by the c-fos technique in hippocampus and somatosensory cortex were analyzed. However, the hippocampal and cortical stimulatory effect detected in vitro could not be demonstrated by these in vivo assays. Altogether, the toxin enhanced the basic excitability of neurons and neuronal networks after direct treatment but there were no effects on the given brain areas after systemic treatment in vivo. Based on the observed in vitro FB1 effects and the lack of data on the penetration of FB1 across the blood-brain barrier, we assume that in vivo consequences of FB1 administration can be more prominent in case of perturbed blood-brain barrier functions.

Coordination of AMPA receptor trafficking by Rab GTPases.

Synaptic connections in the brain are continuously weakened or strengthened in response to changes in neuronal activity. This process, known as synaptic plasticity, is the cellular basis for learning and memory, and is thought to be altered in several neuronal disorders. An important aspect of synaptic plasticity is the tightly controlled trafficking and synaptic targeting of the AMPA-type glutamate receptors, which are the major mediators of fast excitatory transmission in the brain. This review addresses the role of Rab GTPases in AMPA receptor trafficking in neurons under basal conditions and during activity-induced synaptic plasticity, especially during long-term potentiation (LTP) and long-term depression (LTD). We highlight the importance of the tight spatio-temporal control of Rab activity and suggest that this is critical for proper neuronal functions. We also discuss how abnormal AMPA receptor trafficking and malfunctioning of Rabs can lead to neurologic disorders or memory problems.

Alternative classifications of neurons based on physiological properties and synaptic responses, a computational study.

One of the central goals of today's neuroscience is to achieve the conceivably most accurate classification of neuron types in the mammalian brain. As part of this research effort, electrophysiologists commonly utilize current clamp techniques to gain a detailed characterization of the neurons' physiological properties. While this approach has been useful, it is not well understood whether neurons that share physiological properties of a particular phenotype would also operate consistently under the action of natural synaptic inputs. We approached this problem by simulating a biophysically diverse population of model neurons based on 3 generic phenotypes. We exposed the model neurons to two types of stimulation to investigate their voltage responses under conventional current step protocols and under simulated synaptic bombardment. We extracted standard physiological parameters from the voltage responses elicited by current step stimulation and spike arrival times descriptive of the model's firing behavior under synaptic inputs. The biophysical phenotypes could be reliably identified using classification based on the 'static' physiological properties, but not the interspike interval-based parameters. However, the model neurons associated with the biophysically different phenotypes retained cell type specific features in the fine structure of their spike responses that allowed their accurate classification.

Learning to Sort: Few-shot Spike Sorting with Adversarial Representation Learning.

Spike sorting has long been used to obtain activities of single neurons from multi-unit recordings by extracting spikes from continuous data and assigning them to putative neurons. A large body of spike sorting algorithms have been developed that typically project spikes into a low-dimensional feature space and cluster them through iterative computations. However, there is no reached consensus on the optimal feature space or the best way of segmenting spikes into clusters, which often leads to the requirement of human intervention. It is hence desirable to effectively and efficiently utilize human knowledge in spike sorting while keeping a minimum level of manual intervention. Furthermore, the iterative computations that are commonly involved during clustering are inherently slow and hinder real-time processing of large-scale recordings. In this paper, we propose a novel few-shot spike sorting paradigm that employs a deep adversarial representation neural network to learn from a handful of annotated spikes and robustly classify unseen spikes sharing similar properties to the labeled ones. Once trained, the deep neural network can implement a parametric function that encodes analytically the categorical distribution of spike clusters, which can be significantly accelerated by GPUs and support processing hundreds of thousands of recording channels in real time. The paradigm also includes a clustering routine termed DidacticSortto aid users for labeling spikes that will be used to train the deep neural network. We have validated the performance of the proposed paradigm with both synthetic and in vitro datasets.

Comparing the effects of uncoated nanostructured surfaces on primary neurons and astrocytes.

The long-term application of central nervous system implants is currently limited by the negative response of the brain tissue, affecting both the performance of the device and the survival of nearby cells. Topographical modification of implant surfaces mimicking the structure and dimensions of the extracellular matrix may provide a solution to this negative tissue response and has been shown to affect the attachment and behavior of both neurons and astrocytes. In our study, commonly used neural implant materials, silicon, and platinum were tested with or without nanoscale surface modifications. No biological coatings were used in order to only examine the effect of the nanostructuring. We seeded primary mouse astrocytes and hippocampal neurons onto four different surfaces: flat polysilicon, nanostructured polysilicon, and platinum-coated versions of these surfaces. Fluorescent wide-field, confocal, and scanning electron microscopy were used to characterize the attachment, spreading and proliferation of these cell types. In case of astrocytes, we found that both cell number and average cell spreading was significantly larger on platinum, compared to silicon surfaces, while silicon surfaces impeded glial proliferation. Nanostructuring did not have a significant effect on either parameter in astrocytes but influenced the orientation of actin filaments and glial fibrillary acidic protein fibers. Neuronal soma attachment was impaired on metal surfaces while nanostructuring seemed to influence neuronal growth cone morphology, regardless of surface material. Taken together, the type of metals tested had a profound influence on cellular responses, which was only slightly modified by nanopatterning.

Dendritic spine morphology and memory formation depend on postsynaptic Caskin proteins.

CASK-interactive proteins, Caskin1 and Caskin2, are multidomain neuronal scaffold proteins. Recent data from Caskin1 knockout animals indicated only a mild role of Caskin1 in anxiety and pain perception. In this work, we show that deletion of both Caskins leads to severe deficits in novelty recognition and spatial memory. Ultrastructural analyses revealed a reduction in synaptic profiles and dendritic spine areas of CA1 hippocampal pyramidal neurons of double knockout mice. Loss of Caskin proteins impaired LTP induction in hippocampal slices, while miniature EPSCs in dissociated hippocampal cultures appeared to be unaffected. In cultured Caskin knockout hippocampal neurons, overexpressed Caskin1 was enriched in dendritic spine heads and increased the amount of mushroom-shaped dendritic spines. Chemically induced LTP (cLTP) mediated enlargement of spine heads was augmented in the knockout mice and was not influenced by Caskin1. Immunocytochemistry and immunoprecipitation confirmed that Shank2, a master scaffold of the postsynaptic density, and Caskin1 co-localized within the same complex. Phosphorylation of AMPA receptors was specifically altered by Caskin deficiency and was not elevated by cLTP treatment further. Taken together, our results prove a previously unnoticed postsynaptic role of Caskin scaffold proteins and indicate that Caskins influence learning abilities via regulating spine morphology and AMPA receptor localisation.

NMDA receptor NR2B subunit over-expression increases cerebellar granule cell migratory activity.

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C(-/-) mice) or expressing NR2B instead of the NR2C subunit (NR2C-2B mice). BrdU-labeling revealed that over-expression of NR2B increased granule cell translocation in vivo, while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild-type and mutant dissociated cerebellar granule cell cultures isolated from 10-day-old cerebella were similar, but NR2C-2B cultures had elevated level of NR2B subunits and intracellular Ca2+ imaging revealed higher sensitivity towards the addition of NR2B-selective antagonist in vitro. Time-lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C-2B than in wild-type cultures. Our results provide evidence that NR2B-containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression.

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.