Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.

Frequenin (Frq) and its mammalian homologue, Neuronal Calcium Sensor 1 (NCS-1), are calcium-binding proteins, which regulate neurotransmitter release. However, reports are contradictory, and little is known about Frq's cellular mechanisms. The Drosophila nervous system can be used to gain a better understanding of the function of Frq. There are two Frq-encoding genes in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Loss-of-function phenotypes were studied using three different procedures: creating a deletion designed to remove the entire frq1 gene and part of the frq2 gene; using an interfering C-terminal peptide to prevent Frq binding to its intracellular targets; and using RNAi to reduce frq1 and frq2 transcript levels. Deletion of the entire frq1 gene and part of the frq2 gene resulted in impaired neurotransmitter release and enhanced nerve terminal growth. To discriminate chronic from acute loss-of-function effects, the effects of transgenic expression and forward-filling an interfering C-terminal peptide into presynaptic terminals were compared. In both cases, a reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations during development. The chronic treatment also enhanced nerve terminal growth. Conversely, gain-of-function conditions yielded an increase in quantal content and a reduction in nerve terminal growth. Frqs' effects on transmitter output were not due to changes in the number of active zones, nor were they due to changes in the size of the readily releasable pool of vesicles. Oregon Green 488 BAPTA-1 conjugated to 10 kDa Dextran was forward-filled into presynaptic boutons to detect changes in presynaptic Ca2+ signals. Ca2+ responses to presynaptic nerve impulses demonstrated that Frq modulates neurotransmitter release by regulating Ca2+ entry. Gain-of-function phenotypes remained present in a PI4KB null background, demonstrating that Frq's effects were not due to an interaction with PI4KB. All effects seen for all studies were identical for both Frqs, indicating that the two Frq proteins are likely functionally redundant. Overall, Frqs have two distinct functions: one on neurotransmission, primarily by regulating Ca2+ entry, and another on axonal growth and synaptic bouton formation.

Title: “A comparative study of the impact of sustained and intermittent docetaxel chemotherapy in brain in a mouse model” Ji Zhang Master of Science Graduate Department of Pharmaceutical Sciences, University of Toronto November, 2011 Abstract A subset of patients suffers cognitive impairment during or long after chemotherapy. This may result from chemotherapeutic agents crossing the blood brain barrier (BBB). This thesis examined the effects of docetaxel (DTX) on brain toxicity, and the effects of different dosing schedules on brain DTX concentrations and neurotoxicity. Examination of DTX treated mice (total dose of 32mg/kg) revealed appreciable amounts of DTX crossed the BBB after either intermittent (four weekly doses) or sustained (one injection of DTX-PoLigel) administration despite differences in peak drug concentrations and overall exposure profiles. Measurements of autophagy and astrocytes activation not only provided evidence of DTX caused neurotoxicity in the central nervous system, but also revealed a link between dosing schedule and neurotoxicity. Furthermore, the discovery suggested connections between DTX brain exposure, diverse biological events (such as BBB permeability and reactive oxygen species activity), and the microenvironment at synapse-neuron junctions, which should be further explored.

Title: “A comparative study of the impact of sustained and intermittent docetaxel chemotherapy in brain in a mouse model” Ji Zhang Master of Science Graduate Department of Pharmaceutical Sciences, University of Toronto November, 2011 Abstract A subset of patients suffers cognitive impairment during or long after chemotherapy. This may result from chemotherapeutic agents crossing the blood brain barrier (BBB). This thesis examined the effects of docetaxel (DTX) on brain toxicity, and the effects of different dosing schedules on brain DTX concentrations and neurotoxicity. Examination of DTX treated mice (total dose of 32mg/kg) revealed appreciable amounts of DTX crossed the BBB after either intermittent (four weekly doses) or sustained (one injection of DTX-PoLigel) administration despite differences in peak drug concentrations and overall exposure profiles. Measurements of autophagy and astrocytes activation not only provided evidence of DTX caused neurotoxicity in the central nervous system, but also revealed a link between dosing schedule and neurotoxicity. Furthermore, the discovery suggested connections between DTX brain exposure, diverse biological events (such as BBB permeability and reactive oxygen species activity), and the microenvironment at synapse-neuron junctions, which should be further explored.