Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Faculty of Science, Stockholm. / Excerpt from Acta Zoologica. Bd. 2. 1921.

Thesis (Ph. D.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 95-127).

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2000. / Vita. Includes bibliographical references.

Understanding how different regions of the central nervous system (CNS) are affected by genetic insults is critical to advancing the study of CNS pathologies. The cerebellum is one such region which is disproportionately hypoplastic in the majority of cases of CASK gene mutation in humans. CASK is an enigmatic multi-domain scaffolding protein which plays a vital role in organizing protein complexes at the pre-synapse through interactions with both active zone proteins and trans-synaptic adhesion molecules such as liprins-α and neurexins. Mutations in the X-linked CASK gene in humans are largely post-natally lethal in the hemizygous condition and result in microcephaly with pontine and cerebellar hypoplasia (PCH) and also optic nerve hypoplasia (ONH) in heterozygous mutations. Herein, I used various molecular and genetic strategies to uncover the role of the CASK protein in brain function and pathogenesis of cerebellar hypoplasia associated with CASK mutations/deletions. First, using the face- and construct-validated heterozygous CASK knockout (Cask+/-) murine model, I conducted bulk RNA-sequencing and proteomics experiments from whole brain lysates to uncover changes in the Cask+/- brain. RNA-sequencing revealed the majority of changes to be broadly categorized into metabolic, nuclear, synaptic, and extracellular-matrix associated transcripts. Proteomics revealed the majority of changes cluster as synaptic proteins, metabolic proteins, and ribosomal subunits. Thus, absence of CASK in half of brain cells seems to affect synaptic protein content, cell metabolism, and protein homeostasis. Extending these observations, I conducted GFP-trap immunoprecipitation followed by tandem mass spectroscopy to reveal protein complexes in which CASK participates. Commensurate with proteomic changes, CASK was found to complex with synaptic proteins, metabolic proteins, cytoskeletal elements, ribosomal subunits, and protein folding machinery. Next, in order to investigate the pathogenesis of CASK-linked cerebellar hypoplasia, I utilized a human case of early truncation wherein the 27th arginine of CASK is converted to a stop codon. Immunohistochemical analysis of this brain revealed an upregulation of glial fibrillary acidic protein, a common marker for degenerative cell death. To mechanistically test the hypothesis that cerebellar hypoplasia results from cell death rather than developmental failure, I created a murine model wherein CASK is deleted from the majority of cerebellar cells post-development using Cre recombinase driven by the Calb2 promoter. Deleting CASK from all cerebellar granule neurons post-migration indeed leads to degeneration of the cerebellum via massive depletion of granule cells while sparing Purkinje cells. Overall, the cerebellum shrinks by approximately half in cross-sectional area and degeneration is accompanied by a collapsing of the molecular layer and of Purkinje cell dendrites. In addition, cerebellar degeneration presents with a profound locomotor ataxia. In conclusion, CASK seems to be affecting brain energy homeostasis and synaptic connections via interactions with metabolic proteins, synaptic proteins, and protein homeostatic elements. Further, alterations in brain volume associated with CASK-linked disorders is the result of degenerative cell death rather than developmental failure as previously posited. / Doctor of Philosophy / One of the main challenges facing modern neuroscience is the question of how constitutive mutations in genes present in every cell can cause different effects on different parts of the brain. CASK is one such gene which is expressed in every cell of the brain and, when mutated, typically results in an overall smaller brain volume. However, the cerebellum is one region of the brain involved in motor coordination which is disproportionately smaller than the rest of the brain. Through this gene, I investigate here two questions principally: (1) what is the role of the CASK protein in cells? And (2) how is the cerebellum differentially affected? Firstly, I conduct a molecular investigation into what changes in the brain of a mouse model of CASK deletion which recapitulates the majority of human cases found in girls. This genetic model results in half of cells in the body lacking CASK and leads to smaller brain volume with disproportionate reduction in cerebellum size, as in the human subjects. Using a variety of molecular and biochemical methods, I uncover that several classes of proteins are changed in this brain, primarily those associated metabolism and cell-to-cell communication. Further, my experiments indicate that CASK interacts with many of these proteins. Next, I use human cases as well as a novel mouse model to uncover the trajectory of CASK-linked reduction in cerebellar size. The human case indicates molecular signatures of cell death, a surprising finding given that CASK-linked disorders are thought to result from developmental failure. Investigating this mechanistically in a mouse model, I uncover that when CASK is deleted after development, cerebellar cells still die and the cerebellum actually shrinks. Thus, my work herein elucidates potential roles for the CASK molecule in cells and shows, for the first time, that CASK-linked cerebellar size diminishment is degenerative in nature rather than developmental. This degeneration of the cerebellum occurs very early on in infancy and so was missed until now. The most important implication is that a degenerative process could be halted with therapies other than relying exclusively on genetic therapies.

The approaches available for unlocking a neural circuit – deciphering its algorithm’s means and ends – are restricted by the biological characteristics of both the circuit in question and the organism in which it is studied. The cerebellum has long appealed to circuits neuroscientists in this regard because of its simple yet evocative structure and physiology. Decades of efforts to validate theories inspired by its distinctive characteristics have yielded intriguing but highly equivocal results. In particular, the general spirit of David Marr and James Albus’s models of cerebellar involvement in associative learning, now almost 50 years old, continues to shape much research, and yet the resulting data indicates that the Marr-Albus theories cannot, in their original incarnations, be the whole story. In efforts to resolve these mysteries of the cerebellum, researchers have pushed the advantages of its simple circuit even further by studying it in model organisms with complimentary methodological advantages. Much early work for example was conducted in monkeys and humans taking advantage of the mechanically simple and precise oculomotor behaviors at which these foveates excel. Then, as genetic tools entered the scene and became increasingly powerful, neuroscientists began porting what had been learned into mouse, a model system in which these tools can be deployed with great sophistication. This was effective in part because cerebellum is highly conserved across vertebrates so complimentary insights can be made across different model systems. Today genetic prowess has been further augmented by rapid advances in optical methods for visualizing and manipulating genetically targeted components. The promise of these new capabilities provides grounds for exploring additional model organisms with characteristics particularly suited to harnessing the power of modern methodology. In the following chapters I explore the promise and challenges of adding a new organism to the current pantheon of most commonly studied cerebellar model organisms. In chapter 1, I introduce the cerebellar circuit and a sampling of the historically equivocal outcomes met by efforts to test Marr-Albus theories in the context of a classical cerebellar learning paradigm: vestibulo-ocular reflex adaptation. In chapter 2, I detail my efforts to establish a method for population calcium imaging in cerebellar granule cells (GCs) of the weakly electric mormyrid fish, gnathonemus petersii. The unusual anatomical placement of GCs in this organism, directly on the surface of the brain, is ideal for optical methods, which require the ability to illuminate structures of interest. Furthermore, in the mormyrid, GCs play analogous role in two circuits -- the cerebellum and a purely sensory structure, the electrosensory lobe, which has a cerebellum-like structure. This latter circuit is unusually well-characterized and appears to employ a Marr-Albus style associative learning algorithm. This could provide a helpful context for interpreting the purpose of GC processing, shared by this circuit and the cerebellum proper. However, taking advantage of these qualities will require overcoming methodological hurdles presented by imaging in this as-yet not genetically tractable organism. While I was able to load and image evoked transients in these cells, and twice observed spontaneous transient, I did not find a loading method that allowed routine observation of spontaneous levels of activity. In chapter 3, I introduce the larval zebrafish, danio rerio, an organism in which optical and genetic methods are already quite established. The zebrafish is genetically tractable and orders of magnitudes smaller than other vertebrate model systems, making it extremely accessible to optical monitoring and manipulation of neural activity. However, in contrast to the mormyrid, very little is known about the physiology of the cerebellar circuit components in this organism or the behaviors to which they contribute. In chapter 4 I detail my efforts to contribute to this modest foundational knowledge by characterizing the electrophysiological activity of Purkinje cells of larval zebrafish during the optomotor response (OMR)—a behavior with similarities to cerebellar-dependent visual stabilization behaviors that have been studied extensively in mammals. I observe a diversity of structured motor and visual activity that suggests that Purkinje cells could contribute to adjusting swim speed during the OMR and other behaviors. In chapter 5, I outline some of the upfront work that remains before cerebellar researchers are likely to fully harness the power of optical and genetic methods in the zebrafish as well as the types of experiments that may become possible if we do.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Divided attention, or the ability to respond to more than one task simultaneously, is an important skill for navigating complex social, communicative, academic, and professional settings. The purpose of the current study was to understand the association between the volume of the posterior cerebellum and divided attention in individuals with autism spectrum disorders (ASDs) and control participants. It was hypothesized that the ASD group would have worse divided attention abilities and smaller posterior cerebellar volumes compared to the control group. Furthermore, reduced posterior cerebellar volume was expected to be associated with weaker divided attention abilities. Participants were young adult males with high-functioning autism spectrum disorders (n=15) and controls matched for age, handedness, and nonverbal IQ (n=19). Results showed partial support for worse divided attention performance in ASDs and for a positive association between posterior cerebellar volume and divided attention performance. There were no group differences in posterior cerebellar volume, and accounting for intracranial volume did not affect findings. Limitations of the current study and future directions are discussed. / text

No description available.

The voltage-gated potassium channel Kv1.2 is a critical modulator of neuronal physiology, including dendritic excitability, action potential propagation, and neurotransmitter release. However, mechanisms by which Kv1.2 may be regulated in the brain are poorly understood. In heterologous expression systems Kv1.2 is regulated by endocytosis of the channel from the plasma membrane, and this trafficking can be modulated by adenylate cyclase (AC). The goal of this dissertation was to determine whether AC modulated endocytic trafficking of endogenous Kv1.2 occurred in the mammalian nervous system. Within the brain, Kv1.2 is expressed at its highest levels in the cerebellar cortex. Specifically, Kv1.2 is expressed in dendrites of Purkinje cells (PC), the sole efferent neurons of the cerebellar cortex; Kv1.2 is also expressed in axon terminals of Basket cells (BC), which make inhibitory synapses to Purkinje cells. The loss of functional Kv1.2 in PC dendrites or BC axon terminals causes profound changes in the neurophysiology of Purkinje cells, and aberrant loss of Kv1.2 produces cerebellar ataxia. Therefore, the cerebellum offers a brain structure where Kv1.2 is abundant and has known and important roles in synaptic physiology. A candidate regulator of Kv1.2 trafficking in cerebellar synapses is the secretin peptide receptor: the receptor is also located in both PC dendrites and BC axon terminals, and ligand binding to the secretin receptor stimulates AC. Although secretin affects cerebellar neurophysiology and cerebellar dependent behavior, the mechanisms are not well resolved. By cell-surface protein biotinylation and subsequent immunoblot quantitation of secretin treated rat cerebellar slice lysates, secretin was found to decrease cell-surface Kv1.2. This effect could be mimicked by stimulating AC with forskolin, and could be occluded by inhibition of the secretin receptor, AC, or protein kinase A. The secretin receptor stimulated loss of surface Kv1.2 was not accompanied by decreased total Kv1.2 protein levels, but did involve enhanced channel endocytosis. Microscopy studies using two novel independent techniques provided evidence that both BC axon terminals and PC dendrites are sites of AC-stimulated Kv1.2 endocytosis. The physiological significance of secretin mediated suppression of Kv1.2 was supported by collaborative studies which found infusions into the cerebellar cortex of either a toxin that inhibits Kv1.2, or of secretin, enhanced eyeblink conditioning, a form of cerebellar dependent learning, in rats. These studies provided the first evidence that Kv1.2 is regulated by endocytic trafficking in the brain. However, to address the role of that trafficking in synaptic physiology requires knowledge about the determinants of Kv1.2’s endocytic potential, and non-destructive assays to measure Kv1.2 endocytosis in neural circuits. This dissertation therefore concludes with preliminary studies that explore an ancient motif regulating Kv1.2 trafficking, and that discuss a novel dual fluorescent fusion protein reporter of Kv1.2’s subcellular localization.