Adeno-associated viral overexpression of neuroligin 2 in the mouse hippocampus enhances GABAergic synapses and impairs hippocampal-dependent behaviors.

The cell adhesion molecule neuroligin2 (NLGN2) regulates GABAergic synapse development, but its role in neural circuit function in the adult hippocampus is unclear. We investigated GABAergic synapses and hippocampus-dependent behaviors following viral-vector-mediated overexpression of NLGN2. Transducing hippocampal neurons with AAV-NLGN2 increased neuronal expression of NLGN2 and membrane localization of GABAergic postsynaptic proteins gephyrin and GABAARγ2, and presynaptic vesicular GABA transporter protein (VGAT) suggesting trans-synaptic enhancement of GABAergic synapses. In contrast, glutamatergic postsynaptic density protein-95 (PSD-95) and presynaptic vesicular glutamate transporter (VGLUT) protein were unaltered. Moreover, AAV-NLGN2 significantly increased parvalbumin immunoreactive (PV+) synaptic boutons co-localized with postsynaptic gephyrin+ puncta. Furthermore, these changes were demonstrated to lead to cognitive impairments as shown in a battery of hippocampal-dependent mnemonic tasks and social behaviors.

Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model.

Fragile X syndrome (FXS), the most common inherited form of intellectual disability and prevailing known genetic basis of autism, is caused by an expansion in the Fmr1 gene that prevents transcription and translation of fragile X mental retardation protein (FMRP). FMRP binds to and controls translation of mRNAs downstream of metabotropic glutamate receptor (mGluR) activation. Recent work shows that FMRP interacts with the transcript encoding striatal-enriched protein tyrosine phosphatase (STEP; Ptpn5). STEP opposes synaptic strengthening and promotes synaptic weakening by dephosphorylating its substrates, including ERK1/2, p38, Fyn and Pyk2, and subunits of N-methyl-d-aspartate (NMDA) and AMPA receptors. Here, we show that basal levels of STEP are elevated and mGluR-dependent STEP synthesis is absent in Fmr1(KO) mice. We hypothesized that the weakened synaptic strength and behavioral abnormalities reported in FXS may be linked to excess levels of STEP. To test this hypothesis, we reduced or eliminated STEP genetically in Fmr1(KO) mice and assessed mice in a battery of behavioral tests. In addition to attenuating audiogenic seizures and seizure-induced c-Fos activation in the periaqueductal gray, genetically reducing STEP in Fmr1(KO) mice reversed characteristic social abnormalities, including approach, investigation and anxiety. Loss of STEP also corrected select nonsocial anxiety-related behaviors in Fmr1(KO) mice, such as light-side exploration in the light/dark box. Our findings indicate that genetically reducing STEP significantly diminishes seizures and restores select social and nonsocial anxiety-related behaviors in Fmr1(KO) mice, suggesting that strategies to inhibit STEP activity may be effective for treating patients with FXS.

Role of DNA-dependent protein kinase in neuronal survival.

DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme composed of a DNA-binding component called Ku70/80 and a catalytic subunit called DNA-PKcs. Many investigators have utilized DNA-PKcs-deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new-born wild-type and scid mice to investigate the effects of loss of DNA-PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase-3 activation, but treatment with the general caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone did not prevent staurosporine-induced apoptosis. We conclude that a DNA-PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase-independent forms of programmed cell death.

Genetics of central nervous system developmental disorders.

The construction of the nervous system is regulated by genetic and environmental factors. In this article, we have highlighted some of the important molecules and genes that contribute to early stages of CNS development. Future research in the neurosciences will address how genetic and environmental factors interact with each other during brain development and in the mature nervous system.

Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice.

Genetic approaches have provided evidence that DNA end-joining problems serve an essential role in neuronal survival during development of mammalian embryos. In the present study, we tested whether the DNA repair enzyme, DNA dependent protein kinase, plays an important role in the survival of cerebral cortical neurons in mice. DNA-PK is comprised of a DNA-binding subunit called Ku and a catalytic subunit called DNA-PKcs. In mice with the scid mutation, DNA-PKcs is truncated near the kinase domain, which causes loss of kinase activity. We compared the spatial and temporal aspects of neuronal cell death in scid versus isogenic wild-type embryos and found a significant increase in dying cells in scid mice, as assessed by nuclear changes, DNA fragmentation and caspase-3 activity. Additional biochemical and immunocytochemical studies indicated that of several DNA repair enzymes investigated, only PARP was increased in scid mice, possibly in response to elevated DNA strand breaks.

Activation of microglia during developmentally regulated cell death in the cerebral cortex.

Neuronal elimination in the developing CNS is accomplished by an orderly type of cellular suicide called programmed cell death. The principal non-neuronal cells implicated in regulating programmed cell death and subsequent phagocytosis of dying neurons are the brain's macrophage population, the microglia. Little is known about the signaling between microglia and neurons during programmed cell death. However, macrophages in non-neural tissues express receptors for immunoglobulin (IgG) and complement, and these molecules help regulate phagocytosis of dying cells and foreign organisms. Since many of the neurons generated early in CNS development are transient cell types that are immunoreactive for IgG [Upender et al.: J Comp Neurol 1997; 384:271-282], we hypothesized that IgG might alter the phagocytic properties of microglia within the developing nervous system and potentiate engulfment of dying cells. To begin to address this hypothesis, we first asked whether cortical neurons immunoreactive for IgG or calbindin-D28k exhibit morphological evidence of programmed cell death in the cerebral cortex of neonatal rat pups. Secondly, we quantified the incidence of contacts made by microglia on IgG- vs. calbindin-immunoreactive neurons. Thirdly, perturbation experiments were performed to elevate intracortical levels of IgG and the incidence of microglia:neuron contacts were determined. We found that although the nuclei of some IgG-immunoreactive neurons exhibited condensation and fragmentation characteristic of programmed cell death, we did not observe pyknotic calbindin-immunoreactive neurons. IgG-immunoreactive neurons were also more likely to be contacted by microglia than calbindin-immunoreactive neurons. Elevating intracortical levels of IgG experimentally led to a dramatic increase in the expression of microglia complement receptors throughout the cerebral cortex. Taken together, these results suggest that IgG normally present within neuronal subsets in the developing cerebral cortex could serve to locally regulate the expression of complement receptors on microglia.

Immunoglobulin molecules are present in early-generated neuronal populations in the rat cerebral cortex and retina.

Immunoglobulin-like antigens have been identified in neuronal subsets restricted to deeper layers of the developing mammalian cerebral cortex. The pattern is suggestive of selective uptake of immunoglobulin (Ig) from serum or synthesis of Ig or a molecule with Ig-like motifs. To distinguish between these alternatives, biochemical, immunocytochemical, and birthdating analyses were conducted. In neonatal rat cerebral cortex, immunoglobulin-like immunoreactivity was chiefly in subplate neurons, marginal zone neuropil, and processes spanning the cortical plate. Isolated staining was also observed within the ventricular zone. Although most staining in the cortical plate was absent several days after birth, subplate neuron staining persisted until the end of the second postnatal week. Quantitative immunoassays showed that the antigen concentration dropped from 130 ng/mg in cortical cytosol at birth to approximately 80 ng/mg by postnatal day 7 (P7) and remained low thereafter. Two Ig-immunoreactive polypeptides with mobilities similar to heavy and light chains of serum IgG, were identified by Western blotting. The larger band was purified, partially sequenced by Edman degradation, and found to match rat IgG heavy chain. Bromodeoxyuridine birthdating and anti-IgG double-labeling studies showed that most of the Ig-containing cells were early-generated neurons. Outside of the cortex, transient IgG staining was also detected in neurons of the retina and cerebellum. These studies suggest that subplate and other early-generated neurons selectively take up Ig from serum. The IgG may then either be degraded or lost from the central nervous system (CNS) during developmentally regulated cell death.

STEP61: a member of a family of brain-enriched PTPs is localized to the endoplasmic reticulum.

The STEP family of protein tyrosine phosphatases is highly enriched within the CNS. Members of this family are alternatively spliced to produce both transmembrane and cytosolic variants. This manuscript describes the distinctive intracellular distribution and enzymatic activity of the membrane-associated isoform STEP61. Transfection experiments in fibroblasts, as well as subcellular fractionations, sucrose density gradients, immunocytochemical labeling, and electron microscopy in brain tissue, show that STEP61 is an intrinsic membrane protein of striatal neurons and is associated with the endoplasmic reticulum. In addition, structural analysis of the novel N-terminal region of STEP61 reveals several motifs not present in the cytosolic variant STEP46. These include two putative transmembrane domains, two sequences rich in Pro, Glu, Asp, Ser, and Thr (PEST sequences), and two polyproline-rich domains. Like STEP46, STEP61 is enriched in the brain, but the recombinant protein has less enzymatic activity than STEP46. Because STEP46 is contained in its entirety within STEP61 and differs only in the extended N terminus of STEP61, this amino acid sequence is responsible for the association of STEP61 with membrane compartments and may also regulate its enzymatic activity.

Differentiation of glycoconjugates localized to sensory terminals and selected sites in brain.

Distribution of complex carbohydrates in the peripheral and central nervous systems was investigated cytochemically with a lectin that binds specifically to terminal alpha GalNAc and with monoclonal antibodies against carbohydrate epitopes, including glucuronic acid 3-SO4 and chondroitins 6-SO4 and 4-SO4. Comparative staining with these methods differentiated and partially characterized several glycoconjugates in various sites and allowed a comparison of chemical heterogeneity to neural specialization. Distal terminals of sensory neurons concerned with hearing, balance, taste, touch, and sight expressed glucuronyl 3-SO4, which apparently was present in an undefined glycoprotein. Some neurons in sensory nuclei of the brainstem exhibited a similar constituent on their surfaces. Retinal rod outer segments and the cerebellar granular layer possessed masked glucuronyl 3-SO4 that became immunopositive after digestion with chondroitinase ABC and that occurred in chondroitin 6-SO4 and chondroitin 4-SO4, respectively. The surface of neurons in the eighth nerve root and in neighboring nodes of Ranvier stained for unmasked glucuronic acid 3-SO4 and chondroitin 6-SO4. Some neurons of the cerebral cortex expressed unmasked glucuronyl 3-SO4, chondroitin 6-SO4, and terminal alpha GalNAc on their surfaces. Certain cortical neurons and nerve tracts with chondroitin 6-SO4 and terminal alpha GalNAc lacked glucuronyl 3-SO4, and other neurons possessing chondroitin 6-SO4 failed to express either glucuronyl 3-SO4 or terminal alpha GalNAc. Lability of lectin affinity to hyaluronidase suggested the presence of terminal alpha GalNAc in the chondroitin 6-SO4 on cortical neurons. The findings document further the heterogeneity of neural glycoconjugates, expand knowledge about the diversity of neurons with respect to their content of partially characterized glycoconjugates, and link glucuronyl 3-SO4 with or without chondroitin 6-SO4 spatially to sites of active Na+ transport in sensory nerves.

Transient compartmental expression of a family of protein tyrosine phosphatases in the developing striatum.

The expression of a family of intracellular protein tyrosine phosphatases (STEP) was studied in the striatum of rats during ontogeny. Links between the formation of dopamine islands and STEP immunoreactive patches in the striatum were examined since previous work had suggested that STEP isoforms were selectively expressed in dopaminoceptive brain regions. STEP protein and mRNAs were distributed in a patchy manner during the first postnatal week. By 2 weeks, STEP immunoreactivity was homogeneous, indicating that both patch and matrix neurons express STEP by maturity. Two-color immunofluorescent staining was also performed to compare STEP with specific markers for patch and matrix. Tyrosine hydroxylase immunoreactive fibers from the substantia nigra form distinctive dopamine islands in the striatum during late embryonic development, and occupy the sites of future patches [23,37,38,54]. These fiber islands align with STEP immunoreactive neuronal patches during the first two postnatal weeks, suggesting that STEP is a marker for patch neurons in early postnatal development. When STEP's distribution was compared with other markers for patch (substance P) or matrix (calbindin), STEP co-localized with substance P in most striatal neurons on postnatal days 1 through 7. However, STEP was also expressed within a subset of calbindin-positive neurons in the lateral striatum, but not with these neurons elsewhere in the striatum. By adulthood, STEP colocalized with both markers. These results suggest that STEP is expressed first within patch neurons but not matrix, and subsequently within both. The expression of STEP may be triggered by the arrival of striatal afferents or other regulatory factors.

Transient immunoglobulin-like molecules are present in the subplate zone and cerebral cortex during postnatal development.

A monoclonal antibody (mAb SP-1) labels subplate neurons of the cat visual cortex but does not stain the remnants of the subplate neuronal population that comprise the interstitial cells of adult cortical white matter. mAb SP-1 was shown previously to recognize a cytosolic polypeptide of 56 kDa (Naegele et al., 1991). We have now characterized the distribution of SP-1 immunoreactive neurons in the visual cortex and carried out additional biochemical studies at a range of postnatal ages in various tissues. Brain, liver and serum were found to contain the previously identified 56 kDa polypeptide. This polypeptide was also recognized by a cat immunoglobulin antiserum. The epitope recognized by mAb SP-1 was present on cat IgG Fc fragment but not cat IgG Fab fragment. By 4 weeks postnatal, levels of the 56 kDa antigen decreased in cortex and an additional higher molecular weight SP-1 reactive polypeptide of 75 kDa was detected. In the mature cortex, both polypeptides were absent from cytosolic fractions. Immunocytochemical staining comparing the distributions of SP-1 (SP-1+) and anti-IgG (Ig+) immunoreactive neurons showed complete colocalization in subplate neurons beneath primary visual cortex. By 4 weeks, some pyramidal neurons in cortical areas 17 and 18 were weakly positive for SP-1 but negative for IgG. At subsequent ages, the immunoreactive staining became progressively fainter until it was no longer detectable in white or gray matter of adult cat visual cortex.

Cellular and molecular characterization of a brain-enriched protein tyrosine phosphatase.

Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64-66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64-66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64-66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.

Long-term behavioral recovery in parkinsonian rats by an HSV vector expressing tyrosine hydroxylase.

One therapeutic approach to treating Parkinson's disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (L-dopa)-producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine-lesioned rats, used as a model of Parkinson's disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity.

Inverted pyramidal neurons and interneurons in cat cortical subplate zone are labelled by monoclonal antibody SP1.

During development, the subplate zone of the cat neocortex contains neuronal populations with distinct morphological and neurochemical phenotypes. A subset of those are specifically recognized by a mouse monoclonal antibody termed SUBPLATE-1 (SP1), which was generated against tissue homogenates of kitten cortical white matter. SP1 stains cell bodies and proximal dendrites, but rarely distal dendrites, axonal arbors or spines. In order to characterize morphologically the SP1 immunoreactive subplate cell types, we combined SP1 immunohistochemistry with intracellular iontophoretic injections of Lucifer yellow. The majority of double-labelled neurons were inverted pyramids with a single thicker spine-covered dendrite that descended into the white matter and a tuft of thinner spinous dendrites that ascended from the upper somatic pole, but generally remained confined to the white matter. Other double-labelled neurons were multipolar to bitufted, although often equipped with one thicker descending dendrite. In inverted pyramidal cells, the axons originated from the descending dendrite or, more rarely, from the lower portion of the soma, and descended into the white matter. They formed collaterals recurring toward the grey matter. The presence of dendritic spines on double-labelled pyramidal cells and the axonal arborization patterns were two novel features not revealed previously by SP1 immunohistochemistry alone. The inverted pyramidal morphology was typical for double-labelled neurons located in the subplate zone below the apices of the gyri, whereas those located below the flanks or sulci or deep in the white matter often displayed a bitufted or multipolar spinous morphology. A minority of the double-labelled neurons were multipolar with smooth dendrites and locally branching axons. These results suggest that in the cat subplate zone, a majority of the cells expressing the SP1 antigen are spinous, and we termed the spinous subplate cells 'subplate pyramidal neurons'.

Protein tyrosine phosphatases in the nervous system.

Protein tyrosine phosphatases (PTPs) act to oppose the action of tyrosine kinases and serve important roles in regulating levels of phosphotyrosine in cells. Accumulating evidence points to the roles of PTPs in neuronal development and function, as well as neurotransmitter and growth factor receptor signaling cascades. By analogy to the family of tyrosine kinases, there are both receptor-like and intracellular tyrosine phosphatases. A number of these have been identified in the brain and found to be nervous system-enriched. This article describes brain-enriched PTPs, their localization patterns in brain, and speculations regarding their functional roles.

A protein tyrosine phosphatase expressed within dopaminoceptive neurons of the basal ganglia and related structures.

Immunocytochemical and biochemical studies were conducted to characterize a brain-specific protein tyrosine phosphatase, designated STEP for striatal enriched phosphatase. STEP immunoreactivity was most intense in select regions of the CNS receiving a dopaminergic input, and was localized to cell bodies, dendrites, and axonal processes. Western blot analyses of rat brain homogenates revealed a triplet of polypeptides with relative mobilities (M(r)) of 46 kDa, 37 kDa, and 33 kDa enriched within the striatum. Phase separation of protein homogenates by Triton X-114 extraction indicated that this triplet was enriched in soluble but not membrane fractions. Affinity-purified STEP fusion protein exhibited phosphatase activity while a mutated form of the STEP fusion protein (Cys300Ser) showed no demonstrable phosphatase activity.

A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules.

VC1.1 is a monoclonal antibody that stains the surfaces of neuronal subsets in the brain. Previous work showed that VC1.1 recognizes 3 polypeptide bands with molecular weights of 95-105 kDa, 140 kDa and 170 kDa and two high molecular weight proteoglycans with weights of approximately 680 and 650-700 kDa. The heterogeneity and molecular weight range of these bands suggested that VC1.1 might recognize a carbohydrate moiety associated with a family of cell surface molecules. It had been previously demonstrated that a separate monoclonal antibody, HNK-1 also recognized a cell surface associated epitope characterized as a sulfate- and glucuronic acid-containing N-linked carbohydrate. This epitope has been shown to be present on members of the N-CAM adhesion molecule family. In this report, we demonstrate that VC1.1 recognizes an N-linked carbohydrate group that is attached to myelin-associated glycoprotein and N-CAM. Immunocytochemical and biochemical comparisons of VC1.1 and HNK-1 staining in rat and cat brain indicate that these two antibodies probably recognize overlapping, or identical carbohydrate epitopes.

Expression of a unique 56-kDa polypeptide by neurons in the subplate zone of the developing cerebral cortex.

In the mammalian cerebral cortex, neurons destined for layers 2-6 are generated only after the period of genesis for a group of transient neurons that populate the subplate and marginal zones. Although a number of molecular markers for the subplate zone exist, most are also expressed by other cell populations in the cortical plate. To begin to study molecular properties of the subplate, we generated monoclonal antibodies against homogenates of cat cortical subplate zone. One monoclonal antibody, termed subplate 1 (SP1), recognized a polypeptide of 56 kDa. This antigen was strongly expressed within the subplate neurons only during a 3-week period beginning at birth and extending until 3 weeks after birth. From postnatal day 1, the number of SP1-immunoreactive neurons below the visual cortex increased until the end of second postnatal week and then declined thereafter. This period coincides with the period when a majority of the subplate neurons undergo naturally occurring cell death. The antigen was not expressed by subplate neurons surviving in the adult white matter. At the peak of antigen expression, 14% or less of the immunoreactive neurons also coexpressed gamma-aminobutyric acid, somatostatin, or neuropeptide Y. Biochemical and immunocytochemical properties of the SP1 antigen were also compared with the Alz-50 antigen (A68), a marker for dying neurons. On Western blots, SP1- and Alz-50-reactive polypeptides were selectively enriched in cytosolic fractions of kitten cerebral cortex, but each marker recognized different molecular weight polypeptides. In tissue sections many subplate, cortical plate, and layer 1 neurons were Alz-50 immunoreactive. In contrast, a rarer subpopulation of neurons restricted to the subplate was labeled by SP1. We propose that the SP1 antigen is a protein expressed within dying cortical subplate neurons, at the commencement of cell death.

Neuronal subsets express multiple high-molecular-weight cell-surface glycoconjugates defined by monoclonal antibodies Cat-301 and VC1.1.

Cat-301 and VC1.1 are monoclonal antibodies that recognize surface-associated molecules on subsets of mammalian CNS neurons. Earlier work demonstrated that Cat-301 recognizes a 680-kDa chondroitin sulfate proteoglycan (PG). VC1.1 has been shown to recognize 3 polypeptide bands on Western blot analysis; a major band at 95-105 kDa and additional bands at 145 kDa and 170 kDa. In the present report, we show that VC1.1 also reacts with a high-molecular-weight glycoconjugate. Immunoprecipitation experiments and biochemical characterizations indicate that Cat-301 and VC1.1 define at least 3 distinct high-molecular-weight antigens. The VC1.1 antigens react with antikeratan sulfate antibodies, while the Cat-301 antigens do not. By immunodepletion, we show that some VC1.1 antigens are Cat-301 positive, while others are Cat-301 negative. In addition, Cat-301-reactive proteoglycans are heterogeneous with respect to the presence or absence of VC1.1 epitopes. Double-label immunofluorescence studies with these 2 antibodies are consistent with the biochemical results and show that there are 3 classes of immunoreactive neurons in the cat CNS:Cat-301+/VC1.1+, Cat-301-/VC1.1+, and Cat-301+/VC1.1-. These results indicate that structural microheterogeneity exists among Cat-301 and VC1.1 high-molecular-weight glycoconjugates. This heterogeneity may be a reflection of the diverse neuronal phenotypes that are recognized by Cat-301 and VC1.1 in the mammalian CNS.