Karen F. Greif

Glutamate decarboxylases in nonneural cells of rat testis and oviduct : differential expression of GAD65 and GAD67

Abstract: γ‐Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5′‐phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

Postnatal expression of glutamate decarboxylases in developing rat cerebellum.

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD67, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD67 mRNA predominates in the cerebellum. The mRNA for GAD65, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD65 is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD67 mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.

Synaptotagmin-1 promotes the formation of axonal filopodia and branches along the developing axons of forebrain neurons

Synaptotagmin‐1 (syt1) is a Ca2+‐binding protein that functions in regulation of synaptic vesicle exocytosis at the synapse. Syt1 is expressed in many types of neurons well before synaptogenesis begins both in vivo and in vitro. To determine if expression of syt1 has a functional role in neuronal development before synapse formation, we examined the effects of syt1 overexpression and knockdown on the growth and branching of the axons of cultured primary embryonic day 8 chicken forebrain neurons. In vivo these neurons express syt1, and most have not yet extended axons. We present evidence that syt1 plays a role in regulating axon branching, while not regulating overall axon length. To study the effects of overexpression of syt1, we used adenovirus‐mediated infection to introduce a syt1‐YFP construct, or control GFP construct, into neurons. Syt1 levels were reduced using RNA interference. Overexpression of syt1 increased the formation of axonal filopodia and branches. Conversely, knockdown of syt1 decreased the number of axonal filopodia and branches. Time‐lapse analysis of filopodial dynamics in syt1‐overexpressing cells demonstrated that elevation of syt1 levels increased both the frequency of filopodial initiation and their lifespan. Taken together these data indicate that syt1 regulates the formation of axonal filopodia and branches before engaging in its conventional functions at the synapse.

Expression of preproenkephalin mRNA in rat superior cervical ganglion during postnatal development

The number of principal neurons in the rat superior cervical ganglion (SCG) exhibiting enkephalin-peptide immunoreactivity is reported to be limited. To better determine the degree of enkephalinergic phenotype in sympathetic neurons, sections of SCGs from rats aged newborn to adult were processed for in situ hybridization histochemistry, using a [35S]cRNA probe directed against preproenkephalin (PPENK). > 50% of principal ganglion neurons express mRNA for PPENK in adults. Striking variability in labeling intensity is observed. PPENK mRNA is detected in developing ganglia beginning at postnatal days 4-7. Both the number of cells and intensity of labeling increases with postnatal development. These results indicate that expression of PPENK mRNA is more widespread than expression of enkephalin peptides and develops postnatally.

Beyond the cytoskeleton: The emerging role of organelles and membrane remodeling in the regulation of axon collateral branches

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. This article focuses on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches.

Transport of a synaptotagmin-YFP fusion protein in sympathetic neurons during early neurite outgrowth in vitro after transfection in vivo

Developing neurons are engaged in neurite outgrowth as well as the synthesis and transport of proteins involved in synaptic transmission. Very little is known about when transport is established in these rudimentary neurites. We used a novel technique to visualize protein transport during the early hours of neurite outgrowth in culture. Recombinant adenoviruses were used to express a synaptotagmin-YFP fusion protein in the superior cervical ganglia of neonatal rats in vivo and protein transport was examined in neuronal cultures established from the superior cervical ganglions (SCGs). We find that, as early as 4h in culture, synaptotagmin-YFP was present in the cytoplasm, lamellipodia, filopodia and growth cones. Protein expression appeared punctate in neurites at 8h in vitro and is consistent with a vesicular localization. These results indicate that the machinery to transport synapse-specific proteins is functional in rudimentary neurites at this time and indicates that this technique can be used to study early neuronal development.

Changes in expression of a synaptic vesicle antigen in aging sympathetic neurons.

The effects of altering synaptic activity of sympathetic neurons on the expression of a synaptic vesicle protein (p65) were studied by deafferentation of the superior cervical ganglion (SCG) in adult and aged Fischer-344 rats. Levels of p65, an integral membrane protein of synaptic vesicles, were assayed by radioimmunoassay. After deafferentation, a transient increase in p65 levels is observed in the SCG of adult rats. In aged animals, the response to deafferentation is delayed and enhanced, and levels do not drop to values observed in operated adults. After SCG deafferentation, p65 levels in the iris, an SCG target, initially are depressed below control levels; p65 levels return to control values in adult animals after 14 days, but remain depressed in aged animals. In contrast, a transient increase in p65 levels is observed in the pineal of both adult and aged animals. These results suggest that while the aged sympathetic nervous system retains the ability to respond to alterations in synaptic activity, it is unable to reregulate once a response is initiated.

Deafferentation-induced increases in a synaptic vesicle protein in the adult rat superior cervical ganglion are associated with new protein synthesis

Previous studies have shown that deafferentation of the adult rat superior cervical ganglion results in a transient increase in levels of a 65 kDa synaptic vesicle membrane protein (SV). The present study indicates that the observed increase in SV after deafferentation is the result of new protein synthesis. Treatment with cycloheximide, a protein synthesis inhibitor, for 8 h at selected times after surgery produces decreases in SV which are greater than that observed after treatment of unoperated animals. The results suggest that an increased rate of synthesis of this protein is induced by deafferentation. Transsynaptic factors may play important roles in regulation of protein synthesis in sympathetic ganglia.

Age-dependent effects of deafferentation of the rat superior cervical ganglion on expression of P65 (synaptotagmin) during postnatal development

Previous studies have shown that deafferentation of the rat superior cervical ganglion (SCG) alters the levels of p65 (synaptotagmin), a synaptic vesicle integral membrane protein, within the ganglion. Neonatal deafferentation blocks normal postnatal increases in p65, while deafferentation in adult animals produces a transient increase in p65 expression. The present study examines the time course of the shift from the neonatal to adult pattern of response to deafferentation. Neonatal and 7 day old rats showed the neonatal response to deafferentation. Ganglia from rats aged 14 days or older at deafferentation exhibited the transient increase in p65 at 7 days after surgery. The shift from the neonatal to adult response occurs during the second postnatal week. The change in response to deafferentation may be associated with refinement of synaptic function in a manner yet to be determined.