Kantorow, Marc

The striatum, a region of the brain responsible for motor control and reward processing, plays a critical role in various neurological disorders, including Parkinson's disease, Huntington's disease, and addiction. Gnal encodes the heterotrimeric G-protein stimulatory alpha subunit, Gαolf. Gαolf is highly expressed in the striatum, a brain region that is highly relevant to psychosis and psychostimulant drug action. The Gγ7 protein is also enriched in the striatum, where we have previously shown that Gγ7 protein is required at the posttranscriptional level for the hierarchical assembly of the striatal-specific Gαo lfβ2γ7 heterotrimer, which represents the rate-limiting step for cAMP production in striatal D1R and D2R-expressing neurons in the D1 dopamine and A2a adenosine pathways.
Multiple transcripts with variable 3’ UTRs are produced from the Gng7 gene. Previous studies have shown that genes with these characteristics are post-transcriptionally regulated and can be subcellularly localized. Thus, we hypothesized that the γ7 transcripts with variable 3’UTRs act as signaling organizers that regulate the abundance and/or subcellular localization required for preferential assembly and specialized signaling by Golf heterotrimer in the brain. Our findings showed that striatal-enriched γ7 transcripts are post-transcriptionally regulated by virtue of regulatory elements outside of the coding region that bind to its long 3’UTR. These regulatory elements are responsible for translational repression of the γ7 protein. The different length 3’UTRs of the γ7 transcripts 1 and 3 allow for subcellar localization in the nuclei and the neuropil respectively.

During eye lens development the lens receives oxygen from a network of capillaries that comprise of the tunica vasculosa lentis and the anterior pupillary membrane. In development there is regression of this capillaries with the vitreous and aqueous humor, which is the lens only source of oxygen, leaving the lens in low oxygen state. The lens contains a decreasing oxygen gradient from the surface to the core that parallels the differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 alpha (HIF1a), in the regulation of genes required for lens fiber cell differentiation, structure, and transparency. Previous studies by our lab discovered the HIF1a-dependent gene expression patterns of lens genes by utilizing a Multiomics approach that integrated analysis from CUT&RUN, RNA-seq, and ATACseq. Additionally, our lab also established a hypoxia and HIF1a-dependent mechanism for the non-nuclear organelle degradation process required to form mature transparent fiber cells.

Lens differentiation begins with epithelial cells that undergo the process of cellular differentiation and remodelling into fiber cells (Bassnet et al., 2011; Menko 2002; Wride, 2011) that then will undergo terminal remodelling processes to eliminate their cellular organelles to achieve mature lens structure and transparency. We sought to determine if Serine 81, within the minimal essential region (MER) of the BNIP3L protein, is required for organelle elimination. Previous studies have shown that levels of phosphorylated P38 MAPK and ERK ½ peaked in the same region as phosphorylated S81 BNIP3L levels, the equatorial epithelium, where organelle degradation is initiated. The use of specific inhibitors of P38 MAPK (SB203580) or ERK ½ (U0126 or PD99089) and P38 MAPK activator Ansiomycin will be used to determine if P38 MAPK or ERK ½ phosphorylates BNIP3L at S81 to induce mitophagy of mitochondria, endoplasmic reticulum, and Golgi apparatus.

The ocular lens is comprised of an epithelial cell population that undergoes a continuous process of cellular remodeling and differentiation to form elongated transparent fiber cells. This lens differentiation process is hallmarked by the complete elimination of organelles at the center of the lens, elongation of lens fiber cells, and production of lens fiber-cell specific crystallin proteins to form the mature functional structure of the transparent ocular lens. To date, our understanding of the mechanisms that drive the lens differentiation process is incomplete. This dissertation sought to elucidate the potential roles of both hypoxia and epigenetic chromatin remodeling processes as novel regulators of lens differentiation.
The lens lacks a direct blood supply and thus resides in a hypoxic microenvironment. Previous studies revealed the presence of a decreasing oxygen gradient in the region of the lens where cellular remodeling and organelle elimination occur to form mature transparent lens fiber cells. Thus we hypothesized that the hypoxic environment of the lens itself, was required to induce gene expression changes to drive the lens differentiation process. We utilized a multimoics analysis combining CUT&RUN and RNAseq high-throughput sequencing technologies to identify a role for the hypoxia-inducible transcription factor HIF1a as a novel regulator of lens gene expression during lens differentiation.

The lens is responsible for focusing light into the retina. It accomplishes this through its maturation from an epithelial cell into a fiber cell. A large amount of research has been done on cellular differentiation. Nevertheless, we still lack knowledge on many different aspects of differentiation, including a complete theory on the mechanism behind differentiation. Due to the lens’ unique structure and cell types, this is an ideal model for studying differentiation. Our research has shown that αB crystallin, a small heat shock protein, is able to modulate cytochrome C levels and protect the mitochondria under oxidative stress. Also, cytochrome C release is often followed by caspase 3 activation. In addition, research has shown that low levels of caspase 3 activation is essential in driving differentiation. My work examined if αB crystallin could modulate cytochrome C to lower caspase 3 levels to allow for differentiation rather than apoptosis.

αB-crystallin is a small heat-shock chaperone protein (sHSP) required for the homeostasis of multiple tissues including eye lens, retina, heart and brain. Correspondingly, mutation or altered levels of αB-crystallin are associated with multiple degenerative diseases including cataract, retinal degeneration, cardiomyopathy and Lewy body disease. Based on its wide-ranging importance understanding the protective and homeostatic properties of α B-crystallin is critical for understanding degenerative diseases and could lead to the development of therapies to treat these diseases. αB-crystallin is localized to the mitochondria suggesting a direct effect on mitochondrial function. My thesis work has examined those molecular pathways required for translocation of αB-crystallin to the mitochondria and to identify the downstream pathways controlled by mitochondrial translocation of αB-crystallin that could be important for cellular protection and differentiation. My results point to a novel role of αB-crystallin in regulation of key apoptotic pathways that mediate the balance between cell survival and differentiation.

Alzheimer’s disease (AD) has been defined as a type of dementia that causes
problems with memory, thinking, and behavior. AD is characterized by tau tangles and
Aβ plaques in and around neurons, respectively. The impact this disease has on its
victims’ health, both physically and mentally, is unimaginable and the rate of progression
is not expected to decrease any time soon. This threat to our minds encourages the
importance of understanding AD. Amongst the theories as to what bio mechanisms cause
the brain to intertwine is the amyloid cascade hypothesis. The purpose of this thesis is to
review the amyloid cascade hypothesis and discuss treatments which utilize this model.
We also wish to examine social aspects such as loneliness and socioeconomic factors
which are associated with the progression of AD. Research presented provides evidence
that targeting the accumulation of Aβ in the brain will prevent further biochemical
responses to form neurodegenerative pathology. From the collected data, we observe that
therapies targeting the amyloidogenic pathway have received positive feedback in the
medical community. Amongst them, an Aβ synthetic peptide vaccine which made history
in vaccine development due to their responder rate. The impact of social factors such as
loneliness in the advancement of AD is also supported by research. While it is
acknowledged that any neurodegenerative disease is far too complex to narrow its cause
specifically, this thesis provides an association with multiple aspects that can be
understood and applied to future research in this field.

The central premise of this dissertation is that mitochondrial antioxidant enzymes
are essential to lens cell viability by preserving lens cell mitochondria and protecting
and/or repairing lens cell proteins, and two mitochondrial-specific antioxidant enzymes,
Peroxiredoxin 3 (PRDX3) and Methionine sulfoxide reductase A (MsrA), are explored.
In this dissertation, we will examine the expression ofPRDX3 in the human lens, its colocalization
to the lens cell mitochondria, its ability to be induced by H20 2-oxidative
stress, and speculate how PRDX3 function/sf could affect the lens. We will also examine
the reduced levels of MsrA by targeted gene silencing and its effect on reactive oxygen
species production and mitochondrial membrane potential in human lens cells to
determine its role in mitochondrial function in the lens. Lastly, we will examine the
ability of MsrA to repair and restore function to a critical mitochondrial protein,
Cytochrome c. The collective evidence strongly indicates that the loss of mitochondrial-specific enzymes, such as PRDX3 and MsrA, are responsible for increased reactive
oxygen species levels, decreased mitochondrial membrane potential, protein aggregation
and lens cell death, and further indicates that mitochondrial repair, protective, and
reducing systems play key roles in the progression of age-related cataract and other agerelated
diseases.

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.

The lens is a crystallin tissue of the anterior part of the eye that focuses light onto the
retina. Aged-related cataract, which is the result of loss of lens transparency, is the most
common cause of blindness in the world. Being constantly exposed to UV-light, lens is
significantly affected by its UVA spectrum. UV-light exposure has been shown to result
in apoptosis of lens cells which can lead to cataract formation. This suggests the need for
molecular mechanisms to remove apoptotic debris from the lens. In the set of
experiments it was proven that integrin αvβ5-mediated pathway is involved in
phagocytosis of apoptotic cell debris in the ocular lens, thus contributing to its
homeostasis. Additionally, it was shown that exposure to UV-light plays role in cataract
formation by influencing integrin αvβ5-mediated phagocytosis function.