Proteolytic Processing of Neurexins by Presenilins Sustains Synaptic Vesicle Release.

Proteolytic processing of synaptic adhesion components can accommodate the function of synapses to activity-dependent changes. The adhesion system formed by neurexins (Nrxns) and neuroligins (Nlgns) bidirectionally orchestrate the function of presynaptic and postsynaptic terminals. Previous studies have shown that presenilins (PS), components of the gamma-secretase complex frequently mutated in familial Alzheimer's disease, clear from glutamatergic terminals the accumulation of Nrxn C-terminal fragments (Nrxn-CTF) generated by ectodomain shedding. Here, we characterized the synaptic consequences of the proteolytic processing of Nrxns in cultured hippocampal neurons from mice and rats of both sexes. We show that activation of presynaptic Nrxns with postsynaptic Nlgn1 or inhibition of ectodomain shedding in axonal Nrxn1-ß increases presynaptic release at individual terminals, likely reflecting an increase in the number of functional release sites. Importantly, inactivation of PS inhibits presynaptic release downstream of Nrxn activation, leaving synaptic vesicle recruitment unaltered. Glutamate-receptor signaling initiates the activity-dependent generation of Nrxn-CTF, which accumulate at presynaptic terminals lacking PS function. The sole expression of Nrxn-CTF decreases presynaptic release and calcium flux, recapitulating the deficits due to loss of PS function. Our data indicate that inhibition of Nrxn processing by PS is deleterious to glutamatergic function.SIGNIFICANCE STATEMENT To gain insight into the role of presenilins (PS) in excitatory synaptic function, we address the relevance of the proteolytic processing of presynaptic neurexins (Nrxns) in glutamatergic differentiation. Using synaptic fluorescence probes in cultured hippocampal neurons, we report that trans-synaptic activation of Nrxns produces a robust increase in presynaptic calcium levels and neurotransmitter release at individual glutamatergic terminals by a mechanism that depends on normal PS activity. Abnormal accumulation of Nrxn C-terminal fragments resulting from impaired PS activity inhibits presynaptic calcium signal and neurotransmitter release, assigning synaptic defects to Nrxns as a specific PS substrate. These data may provide links into how loss of PS activity inhibits glutamatergic synaptic function in Alzheimer's disease patients.

Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons.

Gene transfer methods for efficient co-expression of exogenous proteins in neurons are crucial tools towards the understanding of the molecular basis of the central nervous system. Lentiviruses are retroviral vectors that can transduce a wide variety of cells including differentiated neurons. In this work, we have generated lentiviral vectors containing dual promoters that allow efficient co-expression of exogenous proteins in neurons. We show that insertion of two copies of a human synapsin promoter/WPRE cassette in a single lentiviral vector directs robust co-expression of cDNAs in cultured neurons, while excluding expression in the surrounding glial cells. Furthermore, insertion of the tetracycline-inducible system (Tet-off) controlled by the synapsin promoter results in tightly regulated expression of EGFP when used as a transgene in cultured neurons. Transduction of primary neurons with this inducible system leads to a 100-fold increase in EGFP mRNA levels in the absence of doxycycline. In transduced cultures, EGFP transcription is inhibited within 24h upon addition of doxycycline. The viral systems we developed here provide neuron-specific and regulated expression mediated by single lentiviral vectors and will prove valuable tools for the study of neuronal function.