Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.

Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosphorylated Akt, which phosphorylates Cx43 on Serine373 and facilitates the forward transport of Cx43 to the plasma membrane, was increased by Gap26 treatment. Microdialysis showed that increased membranous Cx43 causes glutamate release by opening Cx43 hemichannels. Extracellular glutamate concentration was significantly decreased by Gap26 treatment in vivo. Finally, we found that cleaved caspase-3, an apoptosis marker, was attenuated after HI injury by Gap26 treatment. Effects of Gap27 were analogous to those of Gap26. In summary, our findings demonstrate that modulation of Cx43 expression and astroglial function is a potential therapeutic strategy for ischemic brain injury.

Astrocyte-derived ciliary neurotrophic factor (CNTF) promotes adult subventricular zone (SVZ) neurogenesis. We found that focal adhesion kinase (FAK) and JNK, but not ERK or P38, repress CNTF in vitro. Here, we defined the FAK–JNK pathway and its regulation of CNTF in mice, and the related leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), which promote stem cell renewal at the expense of neurogenesis. Intrastriatal injection of FAK inhibitor, FAK14, in adult male C57BL/6 mice reduced pJNK and increased CNTF expression in the SVZ-containing periventricular region. Injection of a JNK inhibitor increased CNTF without affecting LIF and IL-6, and increased SVZ proliferation and neuroblast formation. The JNK inhibitor had no effect in CNTF−/− mice, suggesting that JNK inhibits SVZ neurogenesis by repressing CNTF. Inducible deletion of FAK in astrocytes increased SVZ CNTF and neurogenesis, but not LIF and IL-6. Intrastriatal injection of inhibitors suggested that P38 reduces LIF and IL-6 expression, whereas ERK induces CNTF and LIF. Intrastriatal FAK inhibition increased LIF, possibly through ERK, and IL-6 through another pathway that does not involve P38. Systemic injection of FAK14 also inhibited JNK while increasing CNTF, but did not affect P38 and ERK activation, or LIF and IL-6 expression. Importantly, systemic FAK14 increased SVZ neurogenesis in wild-type C57BL/6 and CNTF+/+ mice, but not in CNTF−/− littermates, indicating that it acts by upregulating CNTF. These data show a surprising differential regulation of related cytokines and identify the FAK–JNK–CNTF pathway as a specific target in astrocytes to promote neurogenesis and possibly neuroprotection in neurological disorders.

No description available.

Disordered communication between cortical neurons and glia underlies many of the characteristics of Fragile X syndrome (FXS), the most common monogenic form of intellectual disability and autism spectrum disorder (ASD). Despite extensive research, no effective treatments exist to comprehensively mitigate ASD- or FXS-related cognitive and motor disabilities, sensory hyperresponsivity, seizures, and other excitation-related symptoms. Glial-glial and glial-neuronal communication can be facilitated by purinergic signalling pathways, which utilize ATP, UTP, and their metabolites to influence both short-term and longer-term activation. The overall objective of this thesis work was to establish whether purinergic signalling is dysregulated within cortical astrocytes derived from the Fmr1 KO mouse model of FXS, and furthermore, to determine whether astrocyte purinergic dysregulations contribute to aberrant Fmr1 KO neuronal-glial interactions. Collectively, these studies provide the first reported evidence that P2Y receptor-driven purinergic signalling is elevated in Fmr1 KO cortical astrocytes, and suggest that this impacts the formation and activity of neuronal circuitry in a manner consistent with FXS symptomatology. Fmr1 KO cortical astrocyte dysregulations included elevated expression of P2Y2 and P2Y6 purinergic receptors, increased intracellular calcium release following P2Y activation, aberrant levels of intracellular purinergic signalling molecules, and increased ectonucleotidase glycosylation. UTP treatment promoted excess Fmr1 KO astrocyte expression and secretion of the synaptogenic protein TSP-1 to potentially influence neuronal connectivity, as well as increased phosphorylation of transcription factor STAT3 to likely drive cortical immune responses. Both exogenous UTP and the presence of Fmr1 KO astrocyte secretions promoted neurite outgrowth, while Fmr1 KO astrocyte-neuron co-cultures demonstrated elevated neuronal burst frequency that was normalized through chronic and selective P2Y2 antagonism. Together, these findings indicate novel and significant astrocyte P2Y-mediated purinergic upregulations within the Fmr1 KO mouse cortex, and suggest that astrocyte purinergic signalling should be further investigated in the search for innovative FXS treatments. / Thesis / Doctor of Philosophy (PhD) / Autism spectrum disorders (ASDs) have become a serious health concern in recent years due to rapidly rising rates of diagnosis. Despite extensive research, there are still no effective treatments for these disorders of brain development. It is therefore important that we study the cellular events contributing to ASDs in order to design new therapeutic strategies. The most common inherited form of ASD is Fragile X syndrome (FXS), which is characterized by cognitive and motor disabilities, sensory hyperresponsivity, attention deficits, hyperactivity, and seizures. Using the Fmr1 knockout (KO) mouse model of FXS, recent research has shown that many of these symptoms are related to disordered communication between brain cells within the cerebral cortex; specifically, between neurons and the helper-like cells known as astrocytes. One form of cellular signalling that supports this communication is known as the purinergic signalling pathway. Collectively, this thesis work is the first to show that purinergic signalling is increased in Fmr1 KO mouse cortical astrocytes and that it impacts FXS neuronal connections. Specifically, Fmr1 KO cortical astrocytes demonstrated increased communication using purinergic signalling, due to greater expression of P2Y2 and P2Y6 purinergic receptors and altered levels of the molecules that stimulate these receptors. Activation of Fmr1 KO astrocyte P2Y receptors promoted expression of the neuronal connection-forming protein TSP-1 and stimulated additional astrocyte signalling pathways. As a result of these changes, when Fmr1 KO neurons were grown in the presence of Fmr1 KO astrocytes, they grew longer extensions and demonstrated greater activity than wildtype controls, in a manner consistent with the excitation-related symptoms of FXS. Selectively targeting P2Y2-driven purinergic pathways with drug treatments corrected this activity, thereby revealing a potential new therapeutic approach for FXS. Understanding excess astrocyte P2Y-driven purinergic communication within the brain may therefore provide a foundation for the future development of new FXS treatments.

No description available.

No description available.

Parkinson's disease (PD) is the second most prevalent age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of dopaminergic neurons within the substantia nigra of the basal ganglia (BG). Motor coordination becomes severely affected over the course of the disease, causing patients to experience tremors at rest, bradykinesia, and body rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and treatment options are limited for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to monogenic variations. Evidence strongly indicates that the majority of PD cases are idiopathic and likely driven due to gene by environmental interactions. Reflective of this idea, recent research efforts have turned to genome-wide association studies (GWAS) to provide indications of gene variations, that while not causative of PD, incur increased risk within patient populations. GWAS findings play a particularly crucial role in neurodegenerative interventions, as early identification of patient risk may allow for preventative therapeutics to delay disease onset or reduce symptom severity. Amongst the many gene variants identified as incurring increased PD risk, single-nucleotide polymorphisms (SNPs) in the loci for CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immunological response and a second as an ectoenzyme that modulates bioenergetic functions. The particular functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede pathological indications. In the brain, CD38 expression is most enriched in astrocytes in BG regions, including substantia nigra, midbrain, and striatum. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/-, and Cd38-/- mice by magnetic-activated cell sorting (MACS) to acquire astrocyte isolates. Numerous differentially expressed genes (DEGs) were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and bioenergetic functions. GO analysis further suggested mitochondrial dysfunction in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered bioenergetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk. / Doctor of Philosophy / Parkinson's disease (PD) is the second most common age-related neurodegenerative disease and currently affects over 8 million people worldwide. The primary features of PD include cognitive, behavioral, and motor function deficits induced primarily by the progressive loss of specialized neurons within the substantia nigra of the basal ganglia (BG) brain region. Motor coordination becomes severely affected over the course of the disease, causing patients to experience body tremors, slowness, and rigidity. The availability of treatment options has increased the quality of life for patients experiencing the early stages of PD; however, there exists no cure and little treatment options for those experiencing severe, advanced disease symptoms. Genetic studies in PD patients have led to the identification of causative genes, but revealed that less than 20% of cases can be attributed to specific, individual variations. Evidence strongly indicates that the majority of PD cases are likely caused by small gene changes that interact with environmental factors. Recent research efforts have turned to genome-wide association studies (GWAS) to identify these small changes, that while not causative of PD, may increase risk within patient populations. GWAS findings play a particularly crucial role in treating neurodegenerative diseases, as early identification of patient risk may allow for preventative therapeutics to slow disease onset or reduce symptom severity. Amongst the many small gene changes identified as increasing PD risk, changes in the gene CD38 that cause reduced gene expression are consistently identified as increasing risk. The cluster of differentiation 38 (CD38) protein serves two major roles: one as a receptor for immune responses and a second as an enzyme that impacts how cells produce energy. The functions of CD38 are highly relevant to neurodegenerative contexts, as changes in central nervous system (CNS) inflammatory status and means of cellular energy production typically precede disease pathology. In the brain, CD38 expression is most enriched in astrocytes, specialized brain cells that supports neurons, in regions affected by PD. However, it is not known how CD38 deficiency may contribute to astrocytic dysfunction and neuropathological features of PD. This dissertation describes how CD38 influences astrocytic gene expression and cellular bioenergetics. Astrocyte RNA was sequenced from the BG of one-year old male Cd38+/+, Cd38+/- (50% CD38 loss), and Cd38-/- (100% CD38 loss) mice by magnetic-activated cell sorting (MACS) to acquire astrocytes. Numerous changes in gene expression were identified in Cd38 Cd38+/- and Cd38-/- astrocytes that relate to regulation of cellular health, responses to stress, and energy functions. Further analysis looking at functions, suggested mitochondrial abnormalities in both Cd38+/- and Cd38-/- astrocytes. In a subsequent set of experiments evaluating mitochondrial function by Seahorse XF96 platform, Cd38+/- and Cd38-/- astrocytes displayed altered energetic function. The results herein demonstrate that astrocytic Cd38 expression regulates cellular function and implicates transcriptional changes associated with the hallmarks of neurodegeneration. These findings serve to provide future direction for studies evaluating the relationship between CD38 function and astrocytes as it relates to neurodegenerative PD risk.

Autism spectrum disorders (ASDs) are a diverse class of neurodevelopmental disorders with various aberrant cellular phenotypes such as dysfunctional neurotransmission and irregular neuronal morphology. ASDs have a broad underlying genetic background with many genes linked to their etiology. UBE3A has been identified as a top gene candidate associated with ASD, and overexpression of UBE3A via copy-number variation confers hallmark ASD behaviors in humans and transgenic mice. Our previous work revealed that synapse formation was negatively affected in the Ube3A-ASD mouse model (Ube3A 2X Tg, or simply “2X Tg”). However, the cellular and molecular mechanisms underlying the synaptic dysregulation remain unknown. We sought to identify a cell-type specific mechanism by which these morphological changes were conferred. We found that selective overexpression of Ube3A in neurons failed to induce changes in dendritic spine maturation. In contrast, overexpression of Ube3A in astrocytes resulted in alterations in spines and synapses. Further, we identified thrombospondin-2 (TSP2), a secreted astrocytic glycoprotein promoting synaptogenesis and spinogenesis, is involved in the defective spine maturation. Ube3A overexpression confers a loss of transcriptional down-regulation of TSP2 in astrocytes, and the medium of astrocyte cultures with Ube3A overexpression was sufficient to trigger spine changes similar to that observed in mixed cultures that globally overexpress Ube3A. Importantly, depletion of TSP2 promoted similar loss of dendritic spine maturation, whereas supplement of TSP2 to 2X Tg astrocyte media was able to rescue the spine defects. Furthermore, overexpression of Ube3A in an astrocyte-specific manner recapitulated aberrant dendritic spine maturation as well as autism-like behaviors displayed in 2X Tg mice. Collectively, these findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels.

No description available.