Summary A delineation of the molecular basis of synapse development is crucial for understanding brain function. Co-cultures of neurons with transfected fibroblastoid cells have been used to demonstrate the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the co-culture assay using custom-made full-length cDNA libraries. Re-isolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is far from complete. We identify LRRTM1 as a novel transmembrane protein capable of inducing presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially-induced clustering of LRRTMs mediates postsynaptic differentiation in dendrites. We generate LRRTM1 -/- mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the recently reported linkage of LRRTM1 to handedness and schizophrenia.

The molecular mechanism underlying the pathogenesis of the majority of cases of sporadic Alzheimer's disease (AD) is unknown. A history of stroke was found to be associated with development of some AD cases, especially in the presence of vascular risk factors. Reduced cerebral perfusion is a common vascular component among AD risk factors, and hypoxia is a direct consequence of hypoperfusion. Previously we showed that expression of the ␤-site ␤-amyloid precursor protein (APP) cleavage enzyme 1 (BACE1) gene BACE1 is tightly controlled at both the transcriptional and translational levels and that increased BACE1 maturation contributes to the AD pathogenesis in Down's syndrome. Here we have identified a functional hypoxiaresponsive element in the BACE1 gene promoter. Hypoxia up-regulated ␤-secretase cleavage of APP and amyloid-␤ protein (A␤) production by increasing BACE1 gene transcription and expression both in vitro and in vivo. Hypoxia treatment markedly increased A␤ deposition and neuritic plaque formation and potentiated the memory deficit in Swedish mutant APP transgenic mice. Taken together, our results clearly demonstrate that hypoxia can facilitate AD pathogenesis, and they provide a molecular mechanism linking vascular factors to AD. Our study suggests that interventions to improve cerebral perfusion may benefit AD patients.hypoxia-inducible factor 1␣ ͉ amyloid-␤ protein ͉ neuritic plaque ͉ memory deficit ͉ transcription D eposition of amyloid-␤ protein (A␤) in the brain is the hallmark of Alzheimer's disease (AD) pathology (1). A␤, the major component of neuritic plaques, is derived from ␤-amyloid precursor protein (APP) after sequential cleavage by ␤-and ␥-secretase. Early-onset familial AD caused by mutations in APP and in the presenilin 1 and 2 genes accounts for only Ϸ5% of total AD cases. The majority of AD cases are sporadic AD with late onset and have no defined cause. The major risk factors for AD include aging, atherosclerosis, diabetes mellitus, stroke, the ApoE 4 polymorphism, and less education. Recent studies have shown that a history of stroke can increase AD prevalence by Ϸ2-fold among elderly patients (2-6). The risk is highest when stroke is concomitant with atherosclerotic vascular risk factors (7). Patients with stroke or cerebral infarction also show poorer cognitive performance and greater severity of clinical dementia (8). Hypoxia is a direct consequence of hypoperfusion, a common vascular component among the AD risk factors, and may play an important role in AD pathogenesis.Oxygen homeostasis is essential for the development and physiology of an organism. Hypoxia-inducible factor 1 (HIF-1) is the principal molecule regulating oxygen homeostasis (9). HIF-1 is a member of the basic helix-loop-helix transcription factor family, and the basic region of the protein binds specifically to the 5Ј-RCGTG hypoxia-responsive element (HRE) in a gene promoter region. HIF-1 contains an oxygen-regulated expression subunit ␣ (HIF-1␣) and a constitutively expressed subunit ␤ (HIF-1␤) (Arnt). HIF-1␣...

Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-kappaB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.

␤-Neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer ␣-neurexins function at both glutamate and ␥-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of ␣-neurexins to neuroligins was recently reported, but the role of the ␣-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three ␣-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. ␣-Neurexins also induce clustering of the GABA A receptor ␥2 subunit. This synapse promoting activity of ␣-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in ␤-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, ␣-neurexinspecific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of ؊S4 to ؉S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, ؉S4 ␤-neurexins and, even more selectively, ␣-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.

Dynamics of GABAergic