Nuclear LIM interactor, a rhombotin and LIM homeodomain interacting protein, is expressed early in neuronal development.

LIM domain-containing transcription factors, including the LIM-only rhombotins and LIM-homeodomain proteins, are crucial for cell fate determination of erythroid and neuronal lineages. The zinc-binding LIM domains mediate protein-protein interactions, and interactions between nuclear LIM proteins and transcription factors with restricted expression patterns have been demonstrated. We have isolated a novel protein, nuclear LIM interactor (NLI), that specifically associates with a single LIM domain in all nuclear LIM proteins tested. NLI is expressed in the nuclei of diverse neuronal cell types and is coexpressed with a target interactor islet-1 (Isl1) during the initial stages of motor neuron differentiation, suggesting the mutual involvement of these proteins in the differentiation process. The broad range of interactions between NLI and LIM-containing transcription factors suggests the utilization of a common mechanism to impart unique cell fate instructions.

Adenovirus-mediated gene delivery into neuronal precursors of the adult mouse brain.

Precursor cells found in the subventricular zone (SVZ) of the adult brain can undergo cell division and migrate long distances before differentiating into mature neurons. We have investigated the possibility of introducing genes stably into this population of cells. Replication-defective adenoviruses were injected into the SVZ of the lateral ventricle of adult mice. The adenoviruses carried a cDNA for the LacZ reporter or the human p75 neurotrophin receptor, for which species-specific antibodies are available. Injection of the viruses into the SVZ led to efficient labeling of neuronal precursors. Two months after viral injection, infected cells were detected in the olfactory bulb, a significant distance from the site of injection. Labeled periglomerular and granular neurons with extensive dendritic arborization were found in the olfactory bulb. These results demonstrate that foreign genes can be efficiently introduced into neuronal precursor cells. Furthermore, adenovirus-directed infection can lead to long-term stable gene expression in progenitor cells found in the adult central nervous system.

Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.

A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

Mnemonic neuronal activity in somatosensory cortex.

Single-unit activity was recorded from the hand areas of the somatosensory cortex of monkeys trained to perform a haptic delayed matching to sample task with objects of identical dimensions but different surface features. During the memory retention period of the task (delay), many units showed sustained firing frequency change, either excitation or inhibition. In some cases, firing during that period was significantly higher after one sample object than after another. These observations indicate the participation of somatosensory neurons not only in the perception but in the short-term memory of tactile stimuli. Neurons most directly implicated in tactile memory are (i) those with object-selective delay activity, (ii) those with nondifferential delay activity but without activity related to preparation for movement, and (iii) those with delay activity in the haptic-haptic delayed matching task but no such activity in a control visuo-haptic delayed matching task. The results indicate that cells in early stages of cortical somatosensory processing participate in haptic short-term memory.

Functional specificity of Hoxa-4 in vertebral patterning lies outside of the homeodomain.

The Hox family of proteins plays a central role in establishing the body plan of a wide range of metazoan organisms. Each member of this family of transcriptional regulators has a distinct functional specificity, yet they bind to similar DNA target sequences through their conserved homeodomain. The mechanisms whereby Hox proteins achieve their diverse specificities in vivo remain undefined. Using the opposing effects of Hoxa-4 and Hoxc-8 in vertebral patterning, we demonstrate by replacing the homeodomain of Hoxa-4 with that of Hoxc-8 that the functional specificity of Hoxa-4 does not track with the homeodomain. These observations provide evidence that other regions of Hox proteins play an important role in mediating functional specificity during mammalian embryogenesis.

Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Low lead levels stunt neuronal growth in a reversible manner.

The developing brain is particularly susceptible to lead toxicity; however, the cellular effects of lead on neuronal development are not well understood. The effect of exposure to nanomolar concentrations of lead on several parameters of the developing retinotectal system of frog tadpoles was tested. Lead severely reduced the area and branchtip number of retinal ganglion cell axon arborizations within the optic tectum at submicromolar concentrations. These effects of lead on neuronal growth are more dramatic and occur at lower exposure levels than previously reported. Lead exposure did not interfere with the development of retinotectal topography. The deficient neuronal growth does not appear to be secondary to impaired synaptic transmission, because concentrations of lead that stunted neuronal growth were lower than those required to block synaptic transmission. Subsequent treatment of lead-exposed animals with the chelating agent 2,3-dimercaptosuccinic acid completely reversed the effect of lead on neuronal growth. These studies indicate that impaired neuronal growth may be responsible in part for lead-induced cognitive deficits and that chelator treatment counteracts this effect.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Polarity of microtubule assemblies during neuronal cell migration.

The active migration of neurons from their sites of origin to their final destinations requires the unidirectional translocation of the nuclei and somatic cytoplasm within the growing leading processes. To explore the cellular machinery underlying this translocation, we determined the polarity of microtubules situated within the leading and trailing processes of migrating cerebellar granule cells in situ. Our analysis reveals that the newly assembled positive ends of the microtubules in the leading process uniformly face the growing tip, while their disintegrating negative ends face the nucleus. In the trailing process, by contrast, microtubule arrays are of mixed polarity. We suggest that the dynamics of slow polymerization in combination with fast disintegration of oriented microtubules create "push" and "pull" forces that contribute to the piston-like saltatory displacement of the nucleus and cytoplasm within the membrane cylinder of the leading process of the migrating neuron.

Expression and analysis of presenilin 1 in a human neuronal system: localization in cell bodies and dendrites.

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or O-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.

Behavioral genetics of thermosensation and hygrosensation in Drosophila.

Whereas temperature and humidity are critical variables affecting physiology, behavior, and evolution, the genetic and neuronal underpinnings of thermosensation and hygrosensation remain poorly understood. We have initiated a behavioral-genetic investigation of these sensory systems in Drosophila. Behavioral tests are described for the rapid screening of mutants defective in thermosensation and hygrosensation. We demonstrate the strong responses of normal flies to temperature and humidity. Two mutants were found with defects in thermosensation, only one of which is also defective in hygrosensation, indicating that they involve different sensory mechanisms. Ablation experiments further separate these sensory systems by showing that thermoreceptors are housed in the third antennal segment, whereas hygroreceptors are located more distally in the antennal arista.