Specific Neuronal Functions Research Articles

Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2.

NeuroD (ND) is a basic helix-loop-helix transcription factor important for neuronal development and survival. By using a yeast two-hybrid screen, we identified two proteins that interact with ND, huntingtin-associated protein 1 (HAP1) and mixed-lineage kinase 2 (MLK2), both of which are known to interact with huntingtin (Htt). Htt is a ubiquitous protein important for neuronal transcription, development, and survival, and loss of its function has been implicated in the pathogenesis of Huntington's disease, a neurodegenerative disorder. However, the mechanism by which Htt exerts its neuron-specific function at the molecular level is unknown. Here we report that Htt interacts with ND via HAP1, and that MLK2 phosphorylates and stimulates the activity of ND. Furthermore, we show that Htt and HAP1 facilitate the activation of ND by MLK2. To our knowledge, ND is the first example of a neuron-specific transcription factor involved in neuronal development and survival whose activity is modulated by Htt. We propose that Htt, together with HAP1, may function as a scaffold for the activation of ND by MLK2.

Receptor Subtypes Involved in the Presynaptic and Postsynaptic Actions of Dopamine on Striatal Interneurons

By stimulating distinct receptor subtypes, dopamine (DA) exerts presynaptic and postsynaptic actions on both large aspiny (LA) cholinergic and fast-spiking (FS) parvalbumin-positive interneurons of the striatum. Lack of receptor- and isoform-specific pharmacological agents, however, has hampered the progress toward a detailed identification of the specific DA receptors involved in these actions. To overcome this issue, in the present study we used four different mutant mice in which the expression of specific DA receptors was ablated. In D1 receptor null mice, D1R-/-, DA dose-dependently depolarized both LA and FS interneurons. Interestingly, SCH 233390 (10 microm), a D1-like (D1 and D5) receptor antagonist, but not l-sulpiride (3-10 microm), a D2-like (D2, D3, D4) receptor blocker, prevented this effect, implying D5 receptors in this action. Accordingly, immunohistochemical analyses in both wild-type and D1R-/- mice confirmed the expression of D5 receptors in both cholinergic and parvalbumin-positive interneurons of the striatum. In mice lacking D2 receptors, D2R-/-, the DA-dependent inhibition of GABA transmission was lost in both interneuron populations. Both isoforms of D2 receptor, D2L and D2S, were very likely involved in this inhibitory action, as revealed by the electrophysiological analysis of the effect of the DA D2-like receptor agonist quinpirole in two distinct mutants lacking D2L receptors and expressing variable contents of D2S receptors. The identification of the receptor subtypes involved in the actions of DA on different populations of striatal cells is essential to understand the circuitry of the basal ganglia and to develop pharmacological strategies able to interfere selectively with specific neuronal functions.

Mutations in dynein link motor neuron degeneration to defects in retrograde transport.

Degenerative disorders of motor neurons include a range of progressive fatal diseases such as amyotrophic lateral sclerosis (ALS), spinal-bulbar muscular atrophy (SBMA), and spinal muscular atrophy (SMA). Although the causative genetic alterations are known for some cases, the molecular basis of many SMA and SBMA-like syndromes and most ALS cases is unknown. Here we show that missense point mutations in the cytoplasmic dynein heavy chain result in progressive motor neuron degeneration in heterozygous mice, and in homozygotes this is accompanied by the formation of Lewy-like inclusion bodies, thus resembling key features of human pathology. These mutations exclusively perturb neuron-specific functions of dynein.

Expression of the survival of motor neuron (SMN) gene in primary neurons and increase in SMN levels by activation of the N-methyl-D-aspartate glutamate receptor.

Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease caused by mutations of the survival of motor neuron (SMN) gene. The SMN protein is expressed ubiquitously as part of a 300-kilodalton multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. During the differentiation process of spinal motor neurons and cerebellar granule cells, the acquisition of mature electrophysiological and molecular properties is linked to the activation of the glutamate receptors of N-methyl-D-aspartate (NMDA) subtype. We have used primary cultures of rat cerebellar granules to study SMN expression during neuronal differentiation in vitro and in response to the activation of the NMDA receptor. We report that the expression of gems, the nuclear structures where SMN concentrates, is developmentally regulated. The highest expression is associated with the cell clustering phase and expression of NMDA receptors. Stimulation of the NMDA receptor induces an increase in gem number and in SMN transcription, through activation of its promoter. These results demonstrate that SMN levels are dependent on synaptic activity, implying that SMN may have important neuron-specific functions downstream of synaptic activation.

Modulating Ca2+ clearance from neurons.

Neurons are exquisitely sensitive to the duration, amplitude and localization of transient increases in intracellular Ca2+ concentration ([Ca2+]i). Modulation of Ca2+ uptake into the mitochondrion and endoplasmic reticulum, and efflux via the plasma membrane Ca2+ pump and Na+/Ca2+ exchange profoundly affect the shape of [Ca2+]i signals. Ca2+ clearance mechanisms are modulated by other signaling pathways, are sensitive to metabolic state and have a memory of the recent history of cell activation. We present here examples of pharmacologic and endogenous regulation of Ca2+ sequestration and efflux in neurons. Ca2+ clearance mechanisms differentially shape [Ca2+]i signals based on their affinity, capacity and location; their modulation alters specific neuronal functions. The increasingly apparent diversity of the molecular entities that make up the [Ca2+]i regulatory system reveals new sites for modulation. Specialized Ca2+ clearance mechanisms participate in unique cellular functions and thus, are important targets for pharmacological and physiological regulation of the neuron.

Modulating CA2+ clearance from neurons

Neurons are exquisitely sensitive to the duration, amplitude and localization of transient increases in intracellular Ca2+ concentration ([Ca2+]i). Modulation of Ca2+ uptake into the mitochondrion and endoplasmic reticulum, and efflux via the plasma membrane Ca2+ pump and Na+/Ca2+ exchange profoundly affect the shape of [Ca2+]i signals. Ca2+ clearance mechanisms are modulated by other signaling pathways, are sensitive to metabolic state and have a memory of the recent history of cell activation. We present here examples of pharmacologic and endogenous regulation of Ca2+ sequestration and efflux in neurons. Ca2+ clearance mechanisms differentially shape [Ca2+]i signals based on their affinity, capacity and location; their modulation alters specific neuronal functions. The increasingly apparent diversity of the molecular entities that make up the [Ca2+]i regulatory system reveals new sites for modulation. Specialized Ca2+ clearance mechanisms participate in unique cellular functions and thus, are important targets for pharmacological and physiological regulation of the neuron.

Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by the degeneration of motor neurons in the spinal cord. The disease is caused by mutations of the survival of motor neuron 1 gene (SMN1), resulting in a reduced production of functional SMN protein. A major question unanswered thus far is why reduced amounts of ubiquitously expressed SMN protein specifically cause the degeneration of motor neurons without affecting other somatic cell types. In a first attempt to address this issue we have investigated the Smn interacting protein 1 (Sip1), with an emphasis on its developmental expression and subcellular distribution in spinal motor neurons in relation to Smn. By confocal immunofluorescence studies we provide evidence that a significant amount of Smn does not co-localize with Sip1 in neurites of motor neurons, indicating that Smn may exert motor neuron-specific functions that are not dependent on Sip1. Sip1 is highly expressed in the spinal cord during early development and expression decreases in parallel with Smn during postnatal development. Strikingly, reduced production of Smn as observed in cell lines derived from SMA patients or in a mouse model for SMA coincides with a simultaneous reduction of Sip1. The finding that expression of Sip1 and Smn is tightly co-regulated, together with the unique localization of Smn in neurites, may help in understanding the motor neuron-specific defects observed in SMA patients.

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma–glioma cell line

Expression of calcium channel α 1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure–function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel α 1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma–glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of ∼7 pS that was identified as T type. A search for calcium channel α 1 subunit messenger RNA in the 140-3 cells with three different tests only revealed α 1C, whereas α 1A–α 1C were present in the parent NG108-15 line. We made a particular effort to search for α 1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (α 1A, α 1B, α 1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.

Raf-1 and B-Raf proteins have similar regional distributions but differential subcellular localization in adult rat brain.

The Raf kinases play an important and specific role in the activation of extracellular signal-regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron-specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B-Raf and Raf-1 proteins are present in most brain areas, whereas A-Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro-caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf-1 is localized mainly in the cytosolic fraction around the nucleus, whereas B-Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B-Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton-insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization.

Expression of androgen-activating enzymes in cultured cells of developing rat brain.

Dihydrotestosterone and estradiol, two active metabolites formed locally in the brain from testosterone, modulate several functions of the developing rat CNS; these compounds derive from the 5 alpha-reduction or the aromatization of the A-ring of the hormone. Also, progesterone and corticosteroids may be 5 alpha-reduced and subsequently 3 alpha-hydroxylated, becoming modulators of specific neuronal functions. Although the aromatase is a single enzyme, two types of 5 alpha-reductase have been cloned, showing peculiar biochemical properties and probably different functions. Therefore, the isoform(s) of the enzyme 5 alpha-reductase(s) present in early stage of brain development have been characterized in primary neuronal and glial cell cultures obtained from the fetal or neonatal rat brain, respectively. Aromatase expression was also studied. The results have shown that in all the brain cells examined type 1 5 alpha-reductase mRNA is expressed. No specific transcript of type 2 5 alpha-reductase is detectable in any of the cell types examined. Finally, the aromatase gene is expressed only in cultured fetal neurons and especially in those derived from the hypothalamic area of the rat embryos. It is interesting that no aromatase mRNA is detectable in mixed glia or in type 1 astrocytes and oligodendrocytes cultured separately.

Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains.

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells.

Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains

Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I). Currently, the PS-I gene product has not been identified and its function remains unknown. Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa. This antigen was also present in immortalized human and mouse neuronal cell cultures. Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction. In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites. Remarkably the relative partition among these three compartments varied dramatically. A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya. In most of the cerebrum, PS-I staining in axons was very weak or undetectable. By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity. Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites. These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function. The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells. © 1996 Wiley-Liss, Inc.

Persistent activation of cAMP-dependent protein kinase by regulated proteolysis suggests a neuron-specific function of the ubiquitin system in Aplysia

In response to the facilitating neurotransmitter serotonin (5-HT), the cAMP-dependent protein kinase (PKA) acquires a special mnemonic characteristic in Aplysia sensory neurons. PKA becomes persistently activated at basal cAMP concentrations owing to a decreased regulatory (R) to catalytic (C) subunit ratio. We previously implicated ubiquitin-mediated proteolysis in this selective loss of R. Here we show that ubiquitin (Ub), Ub-conjugates and proteasomes are present in cell bodies, axon, neuropil and nerve terminals of Aplysia neurons. Because R subunits are not decreased in muscle exposed to 5-HT, comparison of the two tissues provides a tractable approach to determine how the Ub pathway is regulated. We compared the structure of M1, the muscle-specific R isoform, to that of N4, a major neuronal R isoform, to rule out the possibility that the differences in their stability result from differences in structure. We present evidence that N4 and M1 are encoded by identical transcripts; they also behave similarly as protein substrates for the Ub pathway in extracts of the two tissues. Nervous tissue contains 20-times more free Ub, but we present evidence that the susceptibility of R subunits to degradation in neurons relative to muscle results from the greater capacity of neurons to degrade ubiquitinated proteins through the proteasome. Thus, factors that regulate the activity of proteasomes could underlie the enhanced degradation of R subunits in long-term sensitization.

Current Excitement about the Glutamate Receptor Family

Glutamatergic synaptic transmission provides the major excitatory drive to neurons within the mammalian central nervous system, and the consequent activation of glutamate re ceptors participates in a variety of physiological and pathophysiological processes. There are several classes of ligand-gated glutamate receptors that express characteristic elec trophysiological properties. Glutamate receptors are multimeric proteins encoded by a large family of glutamate receptor genes, and individual genes are expressed by restricted populations of cells. One source of the functional diversity of glutamate receptors is the assembly of subunits into hetero-oligomeric complexes. An additional source of diversity comes from splice variants of individual subunits and editing of the messenger RNA se quence of some subunits at critical sites. Glutamate receptor subunit gene expression is dynamically regulated during development and in response to physiological and patho logical stimuli. Particular combinations of glutamate receptor subunits may express elec trophysiological properties that promote neuronal plasticity, whereas other combinations may have neurotoxic effects. The linkage of particular glutamate receptors to specific neuronal functions may provide an avenue for therapeutic intervention. The Neuroscientist 1:60-63, 1995

Widespread constitutive expression of HSP90 messenger RNA in rat brain

The distribution of constitutively expressed mRNA encoding 2 isoforms of heat shock protein 90 (HSP90) was investigated in adult rat brain by in situ hybridization with 35S-labeled cRNA probes. HSP90 mRNA density was estimated by semiquantitative autoradiography and by microscopic analysis. The distribution of HSP90 transcripts was similar for the 2 probes. HSP90 mRNA was widespread but not ubiquitous, and labeling intensity varied among different regions. In particular, HSP90 mRNA was abundant in limbic system-related structures, such as the hippocampus, amygdala, mamillary body, piriform cortex, enthorhinal cortex, bed nucleus of the stria terminalis, medial habenular nucleus, and preoptic hypothalamic nuclei. The highest abundance of mRNA was detected in the Purkinje cell layer of the cerebellum. High abundance was also present in pineal body, choroid plexus, cerebellar granule cell layer, and cranial nerve nuclei. HSP90 mRNA was present in moderate abundance throughout the cortical gray mantle, in the caudate-putamen, most other thalamic and hypothalamic nuclei, the pontine and spinal gray matter, and the anterior lobe of the hypophysis. Weak hybridization was observed in white matter fiber tracts, glial cells, and leptomeninges, and none was observed in endothelial cells and the smooth muscle of the cerebral microvasculature. Our findings suggest that HSP90 gene products may serve constitutive neuron-specific functions in the unstressed brain.

Localization and subcellular distribution of cellular ras gene products in rat brain

Localization and subcellular distribution of the cellular ras gene products (c- ras p21s) in rat brain were studied by immunofluorescence and immunoblotting using a monoclonal antibody recognizing all of Ki-, Ha- and N- ras p21s. In immunohistochemical analysis, strong immunoreactivity for ras p21s was observed in the neuropile of cerebral and cerebellar cortex. On the other hand, the immunoreactivity of the neuronal perikarya and that of white matter were weak and that of non-neuronal cells was undetectable. In subcellular fractionation analysis of cerebrum, c- ras p21s were found mostly in the particulate fractions and almost half of the particulate-bound c- ras p21s were recovered in the P 2 fraction containing myelin, synaptosomes and mitochondria, approximately one-third were in the P 3 fraction containing microsomes, and the rest were in the P 1 fraction containing nuclei and cell debris. In further fractionation of the P 2 fraction, most of c- ras p21s were associated with synaptosomal fraction. In the synaptosomal fraction, c- ras p21s were highly concentrated in the fractions rich in synaptic plasma membranes and were poorly present in the other fractions rich in synaptic vesicles, intrasynaptosomal mitochondria or postsynaptic densities. The content of c- ras p21s of the original homogenate was calculated to be 0.05% of the total protein and c- ras p21s were distributed in the fractions rich in synaptic plasma membranes with approximately 4-fold enrichment over the original homogenate. These results indicate that c- ras p21s are mainly localized in the synaptic plasma membranes and microsomes and suggest that they may participate in some specific neuronal functions at these sites.

The common structure and activities of four subspecies of rat brain protein kinase C family

Elucidation of the complete sequences of four cDNA clones (α, βI, βII, and γ) of the rat brain protein kinase C family has revealed their common structure composed of a single polypeptide chain with four constant (C 1-C 4) and five variable (V 1-V 5) regions. Although these sequences are highly homologous and closely related to one another V 3-, V 4-, and V 5-regions of γ-subspecies are slightly bigger than the corresponding regions of the other three subspecies. The first constant region, C 1, contains a tandem repeat of cysteine-rich sequence (6, total 12 cysteine residues). The third constant region, C 3, has an ATP-binding sequence which is found in many protein kinases. In adult rat whole brain, the relative activities of α-, βI, βII, and γ-subspecies are roughly 16, 8, 55, and 21%, respectively. γ-Subspecies is expressed after birth apparently only in the central nervous tissue, implying its role in the regulation of specific neuronal functions.

A few axonal proteins distinguish ventral spinal cord neurons from dorsal root ganglion neurons.

A series of proteins putatively involved in the generation of axonal diversity was identified. Neurons from ventral spinal cord and dorsal root ganglia were grown in a compartmented cell-culture system which offers separate access to cell somas and axons. The proteins synthesized in the neuronal cell somas and subsequently transported into the axons were selectively analyzed by 2-dimensional gel electrophoresis. The patterns of axonal proteins were substantially less complex than those derived from the proteins of neuronal cell bodies. The structural and functional similarity of axons from different neurons was reflected in a high degree of similarity of the gel pattern of the axonal proteins from sensory ganglia and spinal cord neurons. Each axonal type, however, had several proteins that were markedly less abundant or absent in the other. These neuron-population enriched proteins may be involved in the implementation of neuronal diversity. One of the proteins enriched in dorsal root ganglia axons had previously been found to be expressed with decreased abundance when dorsal root ganglia axons were co-cultured with ventral spinal cord cells under conditions in which synapse formation occurs (P. Sonderegger, M. C. Fishman, M. Bokoum, H. C. Bauer, and P.G. Nelson, 1983, Science [Wash. DC], 221:1294-1297). This protein may be a candidate for a role in growth cone functions, specific for neuronal subsets, such as pathfinding and selective axon fasciculation or the initiation of specific synapses. The methodology presented is thus capable of demonstrating patterns of protein synthesis that distinguish different neuronal subsets. The accessibility of these proteins for structural and functional studies may contribute to the elucidation of neuron-specific functions at the molecular level.

Control of expression of differentiated functions in neuroblastoma cell hybrids.

Cell hybrids have been extensively utilized for gene mapping; more than 50 enzymes and nonenzyme proteins have been assigned to individual human chromosomes. Hybrids have also been used in the study of differentiation; fusions involving mouse or human neuroblastoma cells and various nonneuronal lines resulted in hybrid cells that continued to express neuronspecific functions. The expression of the differentiated state is, however, not an all-or-none phenomenon: One neuronal trait may be evident in such hybrids, in the absence of others. The potential usefulness of the human neuroblastoma hybrids for the assignment of genes involved in the expression of differentiated functions to specific chromosomes is discussed.