Faculty Bibliography

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

RATIONALE: Continuous performance tests (CPTs) are widely used to assess attentional processes in a variety of disorders including Alzheimer's disease and schizophrenia. Common human CPTs require discrimination of sequentially presented, visually patterned 'target' and 'non-target' stimuli at a single location. OBJECTIVES: The aims of this study were to evaluate the performance of three popular mouse strains on a novel rodent touchscreen test (rCPT) designed to be analogous to common human CPT variants and to investigate the effects of donepezil, a cholinesterase inhibitor and putative cognitive enhancer. METHODS: C57BL/6J, DBA/2J and CD1 mice (n = 15-16/strain) were trained to baseline performance using four rCPT training stages. Then, probe tests assessed the effects of parameter changes on task performance: stimulus size, duration, contrast, probability, inter-trial interval or inclusion of flanker distractors. rCPT performance was also evaluated following acute administration of donepezil (0-3 mg/kg, i.p.). RESULTS: C57BL/6J and DBA/2J mice showed similar acquisition rates and final baseline performance following rCPT training. On probe tests, rCPT performance of both strains was sensitive to alteration of visual and/or attentional demands (stimulus size, duration, contrast, rate, flanker distraction). Relative to C57BL/6J, DBA/2J mice exhibited (1) decreasing sensitivity (d') across the 45-min session, (2) reduced performance on probes where the appearance of stimuli or adjacent areas were changed (size, contrast, flanking distractors) and (3) larger dose- and stimulus duration-dependent changes in performance following donepezil administration. In contrast, CD1 mice failed to acquire rCPT (stage 3) and pairwise visual discrimination tasks. CONCLUSIONS: rCPT is a potentially useful translational tool for assessing attention in mice and for detecting the effects of nootropic drugs.

The regulation of organ size is essential to human health and has fascinated biologists for centuries. Key to the growth process is the ability of most organs to integrate organ-extrinsic cues (eg, nutritional status, inflammatory processes) with organ-intrinsic information (eg, genetic programs, local signals) into a growth response that adapts to changing environmental conditions and ensures that the size of an organ is coordinated with the rest of the body. Paired organs such as the vertebrate limbs and the long bones within them are excellent models for studying this type of regulation because it is possible to manipulate one member of the pair and leave the other as an internal control. During development, growth plates at the end of each long bone produce a transient cartilage model that is progressively replaced by bone. Here, we review how proliferation and differentiation of cells within each growth plate are tightly controlled mainly by growth plate-intrinsic mechanisms that are additionally modulated by extrinsic signals. We also discuss the involvement of several signaling hubs in the integration and modulation of growth-related signals and how they could confer remarkable plasticity to the growth plate. Indeed, long bones have a significant ability for "catch-up growth" to attain normal size after a transient growth delay. We propose that the characterization of catch-up growth, in light of recent advances in physiology and cell biology, will provide long sought clues into the molecular mechanisms that underlie organ growth regulation. Importantly, catch-up growth early in life is commonly associated with metabolic disorders in adulthood, and this association is not completely understood. Further elucidation of the molecules and cellular interactions that influence organ size coordination should allow development of novel therapies for human growth disorders that are noninvasive and have minimal side effects.

BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-beta, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression.

A major impeding factor in designing effective therapies against glioblastoma (GBM) is its extensive molecular heterogeneity and the diversity of microenvironmental conditions within any given tumor. To test whether heterogeneity with the GBM stem cell (GSC) population is required to ensure tumor growth in such diverse microenvironments, we used human GBM biospecimens to examine the identity of cells marked by two established GSC markers: CD133 and activation of the Notch pathway. Using primary GBM cultures engineered to express GFP upon activation of Notch signaling, we observed only partial overlap between cells expressing cell surface CD133 and cells with Notch activation (n = 3 specimens), contrary to expectations based on prior literature. To further investigate this finding, we FACS-isolated these cell populations and characterized them. While both CD133+ (CD133 + /Notch-) and Notch+(CD133-/Notch+) cells fulfill GSC criteria, they differ vastly in their transcriptome, metabolic preferences and differentiation capacity, thus giving rise to histologically distinct tumors. CD133+ GSCs have increased expression of hypoxia-regulated and glycolytic genes, and are able to expand under hypoxia by activating anaerobic glycolysis. In contrast, Notch+ GSCs are unable to utilize anaerobic glycolysis under hypoxia, leading to decreased tumorsphere formation ability. While CD133+ GSCs give rise to histologically homogeneous tumors devoid of large tumor vessels, tumors initiated by Notch+ GSCs are marked by large perfusing vessels enveloped by pericytes. Using a lineage tracing system, we showed that pericytes are derived from Notch+ GSCs. In addition, Notch+ cells are able to give rise to all tumor lineages in vitro and in vivo, including CD133 + /Notch- cells, as opposed to Notch- populations, which have restricted differentiation capacity and do not generate Notch+ lineages. Our findings demonstrate that GSC heterogeneity is a mechanism used by tumors to sustain growth in diverse microenvironmental conditions

The function of a neural circuit is shaped by the computations performed by its interneurons, which in many cases are not easily accessible to experimental investigation. Here, we elucidate the transformation of visual signals flowing from the input to the output of the primate retina, using a combination of large-scale multi-electrode recordings from an identified ganglion cell type, visual stimulation targeted at individual cone photoreceptors, and a hierarchical computational model. The results reveal nonlinear subunits in the circuity of OFF midget ganglion cells, which subserve high-resolution vision. The model explains light responses to a variety of stimuli more accurately than a linear model, including stimuli targeted to cones within and across subunits. The recovered model components are consistent with known anatomical organization of midget bipolar interneurons. These results reveal the spatial structure of linear and nonlinear encoding, at the resolution of single cells and at the scale of complete circuits.

Responses of sensory neurons represent stimulus information, but are also influenced by internal state. For example, when monkeys direct their attention to a visual stimulus, the response gain of specific subsets of neurons in visual cortex changes. Here, we develop a functional model of population activity to investigate the structure of this effect. We fit the model to the spiking activity of bilateral neural populations in area V4, recorded while the animal performed a stimulus discrimination task under spatial attention. The model reveals four separate time-varying shared modulatory signals, the dominant two of which each target task-relevant neurons in one hemisphere. In attention-directed conditions, the associated shared modulatory signal decreases in variance. This finding provides an interpretable and parsimonious explanation for previous observations that attention reduces variability and noise correlations of sensory neurons. Finally, the recovered modulatory signals reflect previous reward, and are predictive of choice behavior.

We perceive a stable environment despite the fact that visual information is essentially acquired in a sequence of snapshots separated by saccadic eye movements. The resolution of these snapshots varies-high in the fovea and lower in the periphery-and thus the formation of a stable percept presumably relies on the fusion of information acquired at different resolutions. To test if, and to what extent, foveal and peripheral information are integrated, we examined human orientation-discrimination performance across saccadic eye movements. We found that humans perform best when an oriented target is visible both before (peripherally) and after a saccade (foveally), suggesting that humans integrate the two views. Integration relied on eye movements, as we found no evidence of integration when the target was artificially moved during stationary viewing. Perturbation analysis revealed that humans combine the two views using a weighted sum, with weights assigned based on the relative precision of foveal and peripheral representations, as predicted by ideal observer models. However, our subjects displayed a systematic overweighting of the fovea, relative to the ideal observer, indicating that human integration across saccades is slightly suboptimal.