Cellular signal transduction

The mitochondrion is the powerhouse of the cell. Therefore, it is critical to the homeostasis of the cell that populations of mitochondria that are damaged or in excess are degraded. The process of targeted elimination of damaged or excess mitochondria by autophagy is called mitophagy. In this report, analysis of the mitophagy regulators PINK1/PARKIN and BNIP3L and their roles are assessed in the lens. PARKIN, an E3 ubiquitin ligase, has been shown to play a role in directing damaged mitochondria for degradation. While BNIP3L, an outer mitochondrial membrane protein, increases in expression in response to excess mitochondria and organelle degradation during cellular differentiation. We have shown that PARKIN is both induced and translocates from the cytoplasm to the mitochondria in human epithelial lens cells upon oxidative stress exposure. In addition, our findings also show that overexpression of BNIP3L causes premature clearance of mitochondria and other organelles, while loss of BNIP3L results in lack of clearance. Prior to this work, PARKIN mediated mitophagy had not been shown to act as a protective cellular response to oxidative stress in the lens. This project also resulted in the novel finding that BNIP3L-mediated mitophagy mechanisms are required for targeted organelle degradation in the lens.

Aging is a biological process that has many detrimental effects due to the
accumulation of oxidative damage to key biomolecules due to the action of free
radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative
damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each
form has been shown to reduce a specific enantiomer of bound and free oxidized
methionine. Effects of Msr have yet to be studied in the major developmental
stages of Drosophila melanogaster despite the enzymes elevated expression
during these stages. A developmental timeline was determined for MsrA mutant,
MsrB mutant, and double null mutants against a wild type control. Results show
that the Msr double mutant is delayed approximately 20 hours in the early/mid
third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon
is due to an issue gaining mass.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS.

Neurons are able to maintain membrane potential and synaptic integrity by an
intricate equilibrium of membrane transporter proteins and ion channels. Two
membrane proteins of particular importance in the vertebrate retina are the
excitatory amino acid transporters (EAATs) which are responsible for the reuptake
of glutamate into both glial and neuronal cells and the sodium potassium
chloride cotransporters (NKCCs) that are responsible for the uptake of chloride
ions into the cell. NKCCs are electro-neutral with the uptake of 2 Cl- coupled to
an exchange of a potassium and Na+ ion into the cells. Therefore, there is little
change of cell membrane potential in the action of NKCCs. In this study the
localization and function of EAATs in the distal retina is investigated. Whole cell
patch clamp recordings in lower vertebrate retina have demonstrated that EAAT2
is the main synaptic EAATs in rod photoreceptors and it is localized to the axon terminals. Furthermore, the action of the transporter seems to be modified by
intracellular calcium concentration. There is also evidence that EAAT2 might be
regulated by feedback from the neuron network by glycinergic and GABAergic
mechanisms. The second half of this study investigates expression of NKCCs in
the retina by western blot analysis and quantitative polymerase chain reaction.
There are two forms of NKCCs, NKCC1 and NKCC2. NKCC1 is mostly
expressed in the central nervous system and NKCC2 was thought to only be
expressed in the kidneys. NKCC1 is responsible for the majority of chloride
uptake into neuronal and epithelial cells and NKCC1 is expressed in the distal
retina where photoreceptors synapse on second order horizontal and bipolar
cells. This study found the expression of NKCC1 in the distal retina to be
regulated by temporal light and dark adaptation. Light adaptation increased
phosphorylated NKCC1 expression (the active form of the cotransporter). The
increase in NKCC1 expression during light adaptation was modulated by
dopamine. Specifically, a D1 receptor agonist increased phosphorylated NKCC1
expression. Dopamine is an essential chemical and receptor known for initiating
light adaptation in retina. Finally, an NKCC1 knockout mouse model was
examined and it revealed that both forms of NKCC are expressed in the
vertebrate retina.

Neuronal cell adhesion molecules of L1 family play a critical role in proper nervous system development. Various mutations on human L1-CAM that lead to severe
neurodevelopmental disorders like retardation, spasticity etc. termed under L1 syndrome. The vertebrr their roles in axon pathfinding, neurite extension and cell migration, howeverate L1CAM and its homolog in Drosophila, neuroglian (nrg) have been well studied fo, much less is known about the mechanisms by which they fine tune synaptic connectivity to control the development and maintenance of synaptic connections within neuronal circuits. Here we characterized the essential role of nrg in regulating synaptic structure and function in vivo in a well characterized Drosophila central synapse model neuron, the Giant Fiber (GF) system. Previous studies from our lab revealed that the phosphorylation status of the tyrosine in the Ankyrin binding FIGQY motif in the intracellular domain of Nrg iscrucial for synapse formation of the GF to Tergo-Trochanteral Motor neuron (TTMn) synapse in the GF circuit.
The present work provided us with novel insights into the role of Nrg-Ank interaction in regulating Nrg function during synapse formation and maintenance. By
utilizing a sophisticated Pacman based genomic rescue strategy we have shown that
dynamic regulation of the Neuroglian–Ankyrin interaction is required to coordinate
transsynaptic development in the GF–TTMn synapse. In contrast, the strength of Ankyrin binding directly controls the balance between synapse formation and maintenance at the NMJ.
Human L1 pathological mutations affect different biological processes distinctively
and thus their proper characterization in vivo is essential to understand L1CAM function.
By utilizing nrg14;P[nrg180ΔFIGQY] mutants that have exclusive synaptic defects and the previously characterized nrg849 allele that affected both GF guidance and synaptic function, we were able to analyze pathological L1CAM missense mutations with respect to their effects on guidance and synapse formation in vivo. We found that the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation and not for axon guidance while L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila.

The ubiquitin ligase Highwire is responsible for cell-autonomously promoting
synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects
in synaptic function and extra branching at the axon terminal, corresponding to transient
branching that occur in the course of giant synapse formation during metamorphosis. The
MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor
Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner,
suggesting different molecular mechanisms downstream of the MAP kinase pathway
govern function and pruning. A novel role for Highwire is revealed, regulating the giant
fiber axon’s ability to respond to external cues regulated by Fos. When expression of the
transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline
glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous
manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology.

The classic guidance molecules, Netrin and its receptor Frazzled (Fra), dictate the strength of
synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating
gap junction localization in the pre-synaptic terminal. In Netrin mutant animals the synaptic
coupling between a giant interneuron and the jump motor neuron was weakened. Dye-coupling
between these two neurons was severely compromised or absent. These mutants exhibited
anatomically and physiologically defective synapses between the giant fiber (GF) and
tergotrochanteral motor neuron (TTMn). In cases where Netrin mutants displayed apparently
normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses.
Dye-coupling between the giant fiber and the motor neuron was reduced or eliminated,
suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap
junctions with antibodies to Shaking-B Innexin (ShakB), they were significantly decreased or
absent in the pre-synaptic terminal of the mutant GF. This data is the first to show that Netrin and
Frazzled regulate placement of gap junctions pre-synaptically at a central synapse. In the Drosophila Giant Fiber System, we demonstrate a mechanism that ensures the monoinnervation of two homologous motor neurons by two homologous interneurons. In a scenario where both interneurons could synapse with both motor neuron targets, each interneuron exclusively synapsed with only one target and the circuit functions at normal physiological levels. This innervation pattern depended on the ratio of netrin-to-frazzled expression. When Netrin was over expressed in the system, shifting the ratio in favor of Netrin,
both interneurons synapsed with both target motor neurons and physiological function was reduced. This resulted in the polyinnervationof a single target. In contrast, when Frazzled was over expressed in the system, one interneuron innervated both targets and excluded the remaining interneuron from making any synaptic contact. This resulted in a single interneuron mono-innervating both motor neurons and physiological function was mutant. The orphaned interneuron made no synaptic contact with either motor neuron target. Physiological function was only normal when the Netrin-Frazzled ratio was at endogenous levels and each GF monoinnervated one motor neuron. When we examined the gap junctions at this synapse in experimental animals, there was a significant reduction of gap junction hemichannels in the presynaptic terminal of axons that deviated from normal innervation patterns. While the synapse dyecoupled, the reduction in gap junction hemichannels reduced function in the circuit.

Metastatic cancers are problematic because they spread throughout the body. A crucial step in cancer metastasis is the separation of the cancer cells from their surrounding normal cells. This occurs due to suppression or destruction of cell adhesion molecules such as E-cadherin, occludin, and various claudins. The Snail and Slug transcription factors play a direct role in suppressing these cell adhesion molecules through their SNAG repression domain. We explored the possibility of developing an ELISA diagnostics capable of detecting soluble E-cadherin, occludin, and claudin fragments in the serum of cancer patients. Using several bioinformatics tools, unique extracellular antigenic sequences were identified on claudins-1, 4, 16, occludin, and E-cadherin. These sequences were cloned as GST fusion proteins, expressed, and purified in large quantities to raise antibodies. In parallel, expression profiling of metastatic cancer cell lines was carried out to derive a correlation between Snail-Slug expression and suppression of cell adhesion molecules.

Harman's theory of aging proposes that a buildup of damaging reactive oxygen species (ROS) is one of the primary causes of the deleterious symptoms attributed to aging. Cellular defenses in the form of antioxidants have evolved to combat ROS and reverse damage; one such group is the methionine sulfoxide reductases (Msr), which function to reduce oxidized methionine. MsrA reduces the S enantiomer of methionine sulfoxide, Met-S-(o), while MsrB reduces the R enantiomer, Met-R-(o). The focus of this study was to investigate how the absence of one or both forms of Msr affects locomotion in Drosophila using both traditional genetic mutants and more recently developed RNA interference (RNAi) strains. Results indicate that lack of MsrA does not affect locomotion. However, lack of MsrB drastically reduces rates of locomotion in all age classes. Furthermore, creation of an RNAi line capable of knocking down both MsrA and MsrB in progeny was completed.