Barry B. Kaplan

Genome-wide linkage survey for genetic loci that influence the development of depressive disorders in families with recurrent, early-onset, major depression

In this report, we describe the results of the first genome‐wide linkage survey for genetic loci that influence the development of unipolar Mood Disorders in 81 families identified by individuals with Recurrent, Early‐Onset, Major Depressive Disorder (RE‐MDD). Model‐free linkage analysis was performed using genotypes for 392 highly informative polymorphisms with an average spacing of 9 cM. The highest maximum LOD score observed, 8.19 (genome‐wide adjusted P ≪ 0.0001), occurred for Recurrent Major Depressive Disorder (R‐MDD) at D2S2321 (205 cM), located 121 kb proximal to CREB1. Nineteen chromosomal regions contained linkage peaks that reached genome‐wide statistical significance (genome‐wide adjusted P < 0.05) and ten of these were “highly significant” (adjusted P < 0.001). Six of the 19 linkage peaks were revealed only when the analysis included covariates to control for the effects of sex and linkage to CREB1. Sex‐specific susceptibility loci were common and preferentially affected the vulnerability of women to developing unipolar Mood Disorders. Five loci revealed evidence of interaction with the CREB1 locus in determining susceptibility (epistasis). A systematic candidate gene analysis is presented and potential overlaps of the linkage regions for unipolar Mood Disorders with those reported for other psychiatric disorders are discussed. The findings suggest that genes whose products participate in cellular signaling pathways that converge on CREB, as well as the target genes whose expression they regulate, may also harbor alleles that affect the development of Mood Disorders and related conditions.

MicroRNA-338 Regulates Local Cytochrome c Oxidase IV mRNA Levels and Oxidative Phosphorylation in the Axons of Sympathetic Neurons

MicroRNAs (miRs) are evolutionarily conserved, noncoding RNA molecules of ∼21 nt that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3′UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA. Transfection of precursor miR-338 into the axons of primary sympathetic neurons decreases COXIV mRNA and protein levels and results in a decrease in mitochondrial activity, as measured by the reduction of ATP levels. Conversely, the transfection of synthetic anti-miR oligonucleotides that inhibit miR-338 increases COXIV levels, and results in a significant increase in oxidative phosphorylation and also norepinephrine uptake in the axons. Our results point to a molecular mechanism by which this microRNA participates in the regulation of axonal respiration and function by modulating the levels of COXIV, a protein which plays a key role in the assembly of the mitochondrial cytochrome c oxidase complex IV.

Axonal and presynaptic protein synthesis: new insights into the biology of the neuron.

The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal -- yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins. To dispel this lingering neglect, we now present the wealth of recent observations bearing on this central idea, and consider their impact on our understanding of the biology of the neuron. We demonstrate that extrasomatic translation sites, which are now well recognized in dendrites, are also present in axonal and presynaptic compartments.

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons

microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction. The data revealed stable expression of a number of mature miRNAs that were enriched in the axons and presynaptic nerve terminals. Among the 130 miRNAs identified in the axon, miR-15b, miR-16, miR-204, and miR-221 were found to be highly abundant in distal axons as compared with the cell bodies of primary sympathetic neurons. Moreover, a number of miRNAs encoded by a common primary transcript (pri-miRNA) were differentially expressed in the distal axons, suggesting that there is a differential subcellular transport of miRNAs derived from the same coding region of the genome. Taken together, the data provide an important resource for future studies on the regulation of axonal protein synthesis and the role played by miRNAs in the maintenance of axonal structure and function as well as neuronal growth and development.

Genetic association between reduced P300 amplitude and the DRD2 dopamine receptor A1 allele in children at high risk for alcoholism

BACKGROUND There is evidence that both reduction in P300 amplitude and the presence of the A1 allele are risk markers for alcoholism. We hypothesized that demonstration of a relationship between the marker and the trait in young children who had not begun to drink regularly would provide evidence for dopaminergic mediation of the reduction in P300 often seen among high-risk children. A previous association between the A1 and the P300 amplitude in screened controls supports the hypothesis that this association occurs in the general population. METHODS Children were assessed using both visual and auditory paradigms to elicit event-related potentials (ERPs). The P300 component of the ERP was investigated with respect to the genetic variation of the Taq1A D2 receptor in these children. RESULTS Genetic association between a marker locus (Taq1 A RFLP near the D2 receptor locus) and the amplitude of P300 was found to be present in 58 high-risk children and their relatives (a total of 100 high-risk individuals). CONCLUSIONS A higher proportion of children from alcoholic families may exhibit lower P300 because more of these children carry the A1 allele than is seen in the normal population.

Local Synthesis of Nuclear-Encoded Mitochondrial Proteins in the Presynaptic Nerve Terminal

One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear‐encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl‐CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447–453, 2001. Published 2001 Wiley‐Liss, Inc.

Association of the APOE ε4 allele with clinical subtypes of late life depression

The APOE genotypes of 45 elderly inpatients with major depression were determined to investigate the relationship of this disorder to irreversible dementia in late life. We specifically tested the hypothesis that the frequency of the APOE e 4 allele is elevated in depressed elders with cognitive impairment or psychotic features, subtypes that have been reported to be at increased risk of developing Alzheimers disease (AD). The frequency of e 4 allele was not elevated in the overall group of 45 inpatients and, contrary to our expectation, was not associated with cognitive impairment in this group. In contrast, the e 4 allele frequency for the patients with psychotic features was nearly four times that for the patients without psychotic features and nearly double that of elderly controls. These data suggest that elderly depressed inpatients with cognitive impairment are at risk for developing AD by an e 4-independent pathway, while those with psychotic features are at risk for developing AD by an e 4-dependent pathway. These findings suggest that subtypes of idiopathic major depression in late life may serve as landmarks that distinguish separable pathogenetic pathways to AD.

Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm

Abstract: Axoplasmic RNA from the giant axon of the squid (Loligo pealii)comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/μg of total RNA) suggests that the poly(A)+ RNA population makes up ∼0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments < 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower‐reacting component (moderately abundant and rare sequences). The latter component accounts for ∼56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of ∼2.7 kilobases and 3.1 × 102 kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents ∼0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Effect of chronic cold exposure on tyrosine hydroxylase mRNA in rat adrenal gland

The effect of chronic stress on the levels of tyrosine hydroxylase (TH) RNA in rat adrenal gland was investigated by RNA-DNA hybridization using a cloned TH cDNA probe. Results of dot-blot hybridization experiments and northern analysis demonstrate that exposure of animals to cold for 1 week results in a 4-5-fold increase in the relative abundance of TH mRNA. This increase in TH mRNA level may underlie the increase in adrenal TH activity that is known to occur when rats are exposed to such cold stress.

Protein synthesis in synaptosomes: a proteomics analysis

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre‐synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [35S]methionine and applied two‐dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution. Autoradiographs of blotted two‐dimensional gels revealed de novo synthesis of about 80 different proteins, 18 of which could be matched to silver‐stained gels that were run in parallel. In‐gel digestion of the matched spots and mass spectrometric analyses revealed the identities of various cytosolic enzymes, cytoskeletal proteins, molecular chaperones and nuclear‐encoded mitochondrial proteins. A number of novel proteins (i.e. not matching with database sequences) were also detected. In situ hybridization was employed to confirm the presence of mRNA and rRNA in synaptosomes. Together, our data show that pre‐synaptic endings of squid photoreceptor neurones actively synthesize a wide variety of proteins involved in synaptic functioning, such as transmitter recycling, energy supply and synaptic architecture.