Cellular signal transduction.

Caenorhabditis elegans optionally enter into a dauer diapause phase that results
in a prolonged life in a semi-dormant state. Entry into and recovery from dauer diapause
includes many physical changes in body structure, physiology, and gene expression.
Entry into dauer diapause is regulated by several signaling pathways including
transforming growth factor (TGF-β). Autophagy plays an important role in dauer
formation and recover. During dauer transformation autophagy is up-regulated and may
play a role in remodeling the molecular structure for long term survival during dauer
diapause. This research helps determine the role of autophagy in dauer development and
recovery mediated through the TGF-β signaling pathway. This research also determines
in which tissue autophagy is necessary for dauer formation and recovery through TGF-β signaling. This research is shedding light on the function of autophagy in the TGF-β
signaling pathway, both processes of which have been linked to tumorigenesis, heart
disease and cancer.

Studies have shown that tumor cells are susceptible to pharmacological targeting
of their altered glycolytic metabolism with a variety of compounds that result in
apoptosis. One such compound, 3-bromopyruvate (3-BP), has been shown to eradicate
cancer in an animal model. However, no studies have shown whether the apoptotic
fragments resulting from 3-BP treatment have the capacity to elicit an immunogenic cell
death that activates dendritic cells, the primary antigen presenting cell in the immune
system. Immunogenic cell death is critical to eliciting an effective adaptive immune
response that selectively kills additional target cells and generates immunological
memory. We demonstrated that 3-bromopyruvate induced apoptosis in a number of
different murine breast cancer cell lines, including the highly metastatic 4T1 line. The
dying tumor cells stimulated immature dendritic cells (DCs) of the immortal JAWS II
cell line to produce high levels of the pro-inflammatory cytokine IL-12, and increased their expression of key co-stimulatory molecules CD80 and CD86. The activated
dendritic cells showed increased uptake of fragments from dying tumor cells that
correlated with the increased levels of calreticulin on the surface and release of high
group motility box 1 (HMGB1) of the latter following 3-BP treatment. Additionally, the
anti-phagocytic signal CD47 present on breast cancer cells was reduced by treatment with
3-bromopyruvate when compared to the levels on untreated 4T1 cells. 3-BP treated breast
cancer cells were able to activate dendritic cells through TLR4 signaling. Signaling was
dependent on both the expression of surface calreticulin and on the extracellular release
of high mobility group box 1 protein (HMGB1) during the process of immunogenic cell
death. Killing by 3-BP was compared to mitoxantrone and doxorubicin, among the few
chemotherapeutics that induce immunogenic cell death. 3-BP killing was likewise
compared to camptothecin, a compound that fails to induce immunogenic cell death.
Importantly, 3-BP did not markedly decrease the levels of the key peptide presenting
molecule MHC I on DCs that were co-cultivated with dying tumor cells. Treatment of the
highly aggressive triple negative BT-20 human breast cancer cell line with 3-BP also
induced an immunogenic cell death, activating human dendritic cells in vitro.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and
form excess (and overlapping) postsynaptic connections. Importantly, these excess
connections are refined during circuit maturation so that only the appropriate connections
remain. This synaptic rearrangement phenomenon has been studied extensively in
vertebrates but many of those models involve complex neuronal circuits with multiple
presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila
melanogaster (the giant fiber circuit), we developed tools that enabled us to study the
molecular development of this circuit; which consists of a bilaterally symmetrical pair of
presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each
presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral,
postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both
giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned
their terminals and refined their connectivity such that only a single postsynaptic
connection remained with the ipsilateral target. Furthermore, if we ablated one of the two
giant fibers during development in wildtype animals, the remaining giant fiber often
retained excess connections with the contralateral target that persisted into adulthood.
After demonstrating that the giant fiber circuit was suitable to study synaptic
rearrangement, we investigated two proteins that might mediate this process. First, we
were able to prevent giant fibers from refining their connectivity by knocking out
highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether
overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would
affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or
Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining
excess connections, while exclusively presynaptic (or postsynaptic) expression of either
protein had no effect. We further showed that by simultaneously reducing (Slit-Robo)
midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we
significantly enhanced the proportion of giant fibers that formed excess connections. Our
findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in
refining synaptic circuits and shaping giant fiber circuit connectivity.

Ischemic stroke has a multiplicity of pathophysiological mechanisms.
Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that
exerts a diverse range of neuroprotection against ischemic stroke. Several lines of
evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell
death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to
10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and
250ng/ml) for 24 hours. Cell viability, expression of the G-CSF receptor and expression
level of CHOP were assessed in vitro. Sprague-Dawley rats were subjected to middle
cerebral artery occlusion (MCAO). Rats were subcutaneously injected with G-CSF (n=
15; 50ug/kg body weight) 24 hours post-MCAO for 4 days. Vehicle treated rats were
administered 5% dextrose for 1 day (n=4) or 4 days (n=16). Sham-operated rats (n=9)
were not subjected to MCAO. Neurological deficit and infarct volume were measured while expression levels of pAKT, Bcl2, Bax, Bak, cleaved caspase-3, GRP78, ATF4,
ATF6, p-p38MAPK, pJNK, CHOP and HSP27 were analyzed by western blotting. In
vitro G-CSF receptor was expressed on undifferentiated PC12 cell, and an optimal dose
of 50 ng/ml G-CSF significantly protected these cells against glutamate-induced
cytotoxicity (P < 0.05). G-CSF significantly down-regulated (P < 0.01) the ER stressinduced
pro-apoptotic marker CHOP in vitro. In vivo, G-CSF reduced infarct volume to
50% while significantly improved neurological deficit compared to vehicle rats. G-CSF
significantly (P < 0.05) up-regulated pro-survival proteins pAKT and Bcl2 while downregulating
pro-apoptotic proteins Bax, Bak and cleaved caspase 3 in the ischemic brain.
It also significantly (P < 0.05) downregulated the ER intraluminal stress sensor GRP78,
proteins of ER stress induced intracellular pathway; ATF4, ATF6, p-p38MAPK, pJNK
and the ER stress induced apoptotic marker CHOP, which suggests that ER stress is
being ameliorated by G-CSF treatment. G-CSF also reduced the level of HSP27,
providing additional evidence of cellular stress reduction. G-CSF treatment increased
cell survival by attenuating both general pro-apoptotic proteins and specific effector
proteins in the ER stress induced apoptotic pathways. Our data has provided new insight
into the anti-apoptotic mechanism of G-CSF, especially as it relates to ER stress induced
apoptosis in ischemia.

The inevitable aging process can be partially attributed to the accumulation of
oxidative damage that results from the action of free radicals. Methionine sulfoxide
reductases (Msr) are a class of enzymes that repair oxidized methionine residues. The
two known forms of Msr are MsrA and MsrB which reduce the R- and S- enantiomers of
methionine sulfoxide, respectively. Our lab has created the first genetic animal model
that is fully deficient for any Msr activity. Previously our lab showed that these animals
exhibit a 20 hour delay in development of the third instar larvae (unpublished data). My
studies have further shown that the prolonged third-instar stage is due to a reduced
growth rate associated with slower food intake and a markedly slower motility. These
Msr-deficient animals also exhibit decreased egg-laying that can be attributed to a lack of
female receptivity to mating.

The vertebrate eye lens functions to focus light onto the retina to produce vision.
The lens is composed of an anterior monolayer of cuboidal epithelial cells that overlie a
core of organelle free fiber cells. The lens develops and grows throughout life by the
successive layering of lens fiber cells via their differentiation from lens epithelial cells.
Lens developmental defect and damage to the lens are associated with cataract formation,
an opacity of the lens that is a leading cause of visual impairment worldwide. The only
treatment to date for cataract is by surgery. Elucidating those molecules and mechanisms
that regulate the development and lifelong protection of the lens is critical toward the
development of future therapies to prevent or treat cataract. To determine those
molecules and mechanisms that may be important for these lens requirements we
employed high-throughput RNA sequencing of microdissected differentiation statespecific
lens cells to identify an extensive range of transcripts encoding proteins expressed by these functionally distinct cell types. Using this data, we identified
differentiation state-specific molecules that regulate mitochondrial populations between
lens epithelial cells that require the maintenance of a functional population of
mitochondria and lens fiber cells that must eliminate their mitochondria for their
maturation. In addition, we discovered a novel mechanism for how lens epithelial cells
clear apoptotic cell debris that could arise from damage to the lens and found that UVlight
likely compromises this system. Moreover, the data herein provide a framework to
determine novel lens cell differentiation state-specific mechanisms. Future studies are
required to determine the requirements of the identified molecules and mechanisms
during lens development, lens defense against damage, and cataract formation.

Establishing appropriate animal models for the study of human memory is
paramount to the development of memory disorder treatments. Damage to the
hippocampus, a medial temporal lobe brain structure, has been implicated in the memory
loss associated with Alzheimer’s disease and other dementias. In humans, the role of the
hippocampus is largely defined; yet, its role in rodents is much less clear due to
conflicting findings. To investigate these discrepancies, an extensive review of the rodent
literature was conducted, with a focus on studies that used the Novel Object Recognition
(NOR) paradigm for testing. The total amount of time the objects were explored during
training and the delay imposed between training and testing seemed to determine
hippocampal recruitment in rodents. Male C57BL/6J mice were implanted with bilateral
dorsal CA1 guide cannulae to allow for the inactivation of the hippocampus at discrete
time points in the task. The results suggest that the rodent hippocampus is crucial to the
encoding, consolidation and retrieval of object memory. Next, it was determined that there is a delay-dependent involvement of the hippocampus in object memory, implying
that other structures may be supporting the memory prior to the recruitment of
hippocampus. In addition, when the context memory and object memory could be further
dissociated, by altering the task design, the results imply a necessary role for the
hippocampus in the object memory, irrespective of context. Also, making the task more
perceptually demanding, by requiring the mice to perform a two-dimensional to three-dimensional
association between stimuli, engaged the hippocampus. Then, in the
traditional NOR task, long and short training exploration times were imposed to
determine brain region activity for weak and strong object memory. The inactivation and
immunohistochemistry findings imply weak object memory is perirhinal cortex
dependent, while strong object memory is hippocampal-dependent. Taken together, the
findings suggest that mice, like humans, process object memory on a continuum from
weak to strong, recruiting the hippocampus conditionally for strong familiarity.
Confirming this functional similarity between the rodent and human object memory
systems could be beneficial for future studies investigating memory disorders.

Neuronal circuit output is dependent on the embedded synapses’ precise
regulation of Ca2+ mediated release of neurotransmitter filled synaptic vesicles (SVs) in
response to action potential (AP) depolarizations. A key determinant of SV release is the
specific expression, organization, and abundance of voltage gated calcium channel
(VGCC) subtypes at presynaptic active zones (AZs). In particular, the relative distance
that SVs are coupled to VGCCs at AZs results in two different modes of SV release that
dramatically impacts synapse release probability and ultimately the neuronal circuit
output. They are: “Ca2+ microdomain,” SV release due to cooperative action of many
loosely coupled VGCCs to SVs, or “Ca2+ nanodomain,” SV release due to fewer more
tightly coupled VGCCs to SVs. VGCCs are multi-subunit complexes with the pore
forming a1 subunit (Cav2.1), the critical determinant of the VGCC subtype kinetics,
abundance, and organization at the AZ. Although in central synapses Cav2.2 and Cav2.1 mediate synchronous transmitter release, neurons express multiple VGCC subtypes with
differential expression patterns between the cell body and the pre-synapse. The calyx of
Held, a giant axosomatic glutamatergic presynaptic terminal that arises from the globular
bushy cells (GBC) in the cochlear nucleus, exclusively uses Cav2.1 VGCCs to support
the early stages of auditory processing. Due to its experimental accessibility the calyx
provides unparalleled opportunities to gain mechanistic insights into Cav2.1 expression,
organization, and SV release modes at the presynaptic terminal. Although many
molecules are implicated in mediating Cav2.1 expression and SV to VGCC coupling
through multiple binding domains on the C-terminus of the Cav2.1 a1 subunit, the
underlying fundamental molecular mechanisms remain poorly defined. Here, using viral
vector mediated approaches in combination with Cav2.1 conditional knock out transgenic
mice, we demonstrate that that there a two independent pathways that control Cav2.1
expression and SV to VGCC coupling at the calyx of Held. These implications for the
regulation of synaptic transmission in CNS synapses are discussed.

Changes in synaptic strength underlie synaptic plasticity, the cellular substrate for learning and memory. Disruptions in the mechanisms that regulate synaptic strength closely link to many developmental, neurodegenerative and neurological disorders. Release site probability (PAZ) and active zone number (N) are two important presynaptic determinants of synaptic strength; yet, little is known about the processes that establish the balance between N and PAZ at any synapse. Furthermore, it is not known how PAZ and N are rebalanced during synaptic homeostasis to accomplish circuit stability. To address this knowledge gap, we adapted a neurophysiological experimental system consisting of two functionally differentiated glutamatergic motor neurons (MNs) innervating the same target. Average PAZ varied between nerve terminals, motivating us to explore benefits for high and low PAZ, respectively. We speculated that high PAZ confers high-energy efficiency. To test the hypothesis, electrophysiological and ultrastructural measurements were made. The terminal with the highest PAZ released more neurotransmitter but it did so with the least total energetic cost. An analytical model was built to further explore functional and structural aspects in optimizing energy efficiency. The model supported that energy efficiency optimization requires high PAZ. However, terminals with low PAZ were better able to sustain neurotransmitter release. We suggest that tension between energy efficiency and stamina sets PAZ and thus determines synaptic strength. To test the hypothesis that nerve terminals regulate PAZ rather than N to maintain synaptic strength, we induced sustained synaptic homeostasis at the nerve terminals. Ca2+ imaging revealed that terminals of the MN innervating only one muscle fiber utilized greater Ca2+ influx to achieve compensatory neurotransmitter release. In contrast, morphological measurements revealed that terminals of the MN inner vating multiple postsynaptic targets utilized an increase in N to achieve compensatory neurotransmitter release, but this only occurred at the terminal of the affected postsynaptic target. In conclusion, this dissertation provides several novel insights into a prominent question in neuroscience: how is synaptic strength established and maintained. The work indicates that tension exists between energy efficiency and stamina in neurotransmitter release likely influences PAZ. Furthermore, PAZ and N are rebalanced differently between terminals during synaptic homeostasis.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2), which has been shown to play a role in axon
outgrowth and guidance of embryonic motorneurons, as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we
characterized the developmental role of PTP69D in the giant fiber (GF) neurons; two
interneurons in the central nervous system (CNS) that control the escape response of the fly. In addition to guidance and targeting functions, our studies reveal an additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of
phosphatase activity in catalytic domain (Cat1) proximal to the transmembrane domain
did not affect axon guidance or targeting but resulted in stunted terminal growth of the
GFs. Cell autonomous rescue and knockdown experiments demonstrated a function for
PTP69D in the GFs, but not its postsynaptic target neurons. In addition,complementation studies and structure-function analyses revealed that for GF terminal growth, Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domain but not the fibronectin type III repeats nor the membrane proximal region. In contrast, the fibronectin type III repeats, but not the immunoglobulin domains, were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function during earlier developmental processes.