Leigh syndrome (LS), the most common type of pediatric mitochondrial disease, has been associated with loss-of-function mutations in genes that encode for proteins in complex 1 of the electron transport chain. Mutations in a gene called NADH dehydrogenase (ubiquinone) iron sulfur protein 4 (NDUFS4) are linked to LS in several groups of patients. Mice carrying a whole body or central nervous system-specific deletion of this gene develop several symptoms reminiscent of those found in humans. Recent studies in our lab showed that knockout (KO) of Ndufs4 in GABAergic interneurons are the key driver of seizures in the mouse model. In this study, we investigated which subtypes of interneurons are the leading cause of epilepsy. We focused on two major subtypes of GABAergic interneurons: interneurons that express parvalbumin (PV), a high-affinity calcium binding protein, and interneurons that express somatostatin (SST), a neuropeptide. PV interneurons make up 40% of GABAergic cells and have faster spike firing patterns when compared to SST cells. We hypothesize that PV neurons will contribute more to seizure susceptibility than SST interneurons because mitochondrial dysfunction is known to affect cells with high firing rates due to their high energy demand. To test our hypothesis, we generated mice with Ndufs4 KO restricted to PV or SST interneurons using LoxP Cre technology. We evaluated the general health of mice and tested their susceptibility to thermal seizures through behavioral checks and thermal induction tests. We found that Ndufs4 KO in both PV and SST interneurons led to seizure phenotypes. However, this gene KO did not have any detectable effect on body weight, motor activity, breathing patterns, physical appearance, and limb extension. A better understanding of mechanisms underlying epileptic seizures will lead to the development of effective treatments for LS-related epilepsy.

The hypoxia response pathway, induced by genetic activation or by decreasing oxygen available, has been shown to extend the lifespan of C. elegans. A previous experiment conducted in our lab compared the transcriptomes of worms treated with normoxia, continuous hypoxia, and intermittent hypoxia therapy (IHT). This study showed that IHT doubles lifespan in C. elegans and was partially controlled by the enzyme inositol polyphosphate multikinase (IPMK-1), which suppresses some of the lifespan extension benefits of IHT. To further explore the genetic basis for the effect of IPMK-1 on IHT, we performed a forward genetic suppressor screen on the IPMK-1 animals. IPMK-1 animals die at elevated temperature so we mutagenized IPMK-1(sea9) worms and selected animals from the F2 generation that reached adulthood at 26.5^oC. Identifying the genetic changes in these suppressors will tell us more about the control of IHT and how it promotes longevity.

A challenge that teachers often face is creating a curriculum and environment that is conducive to learning, more broadly defined as the ability to recall information and concepts. The goal of this research is to provide a framework for teachers, in particular Argentine tango teachers, to combat that challenge in a way that is coherent with how memory is stored in the brain. Three observations were collected: one of a 10-week tango course taught by one professor twice weekly at the University of Washington, and two others were drop-in classes of three local tango teachers in the Seattle area. Three practices to be used in combination were found to strengthen curriculum: Spaced Practice—where students “space out” what they learn or revisit a subject or movement over a period of time, versus cramming information all at once; Varied Practice—where information is recalled and applied in various ways (engaging all the senses and emotions); and Interleaved Practice—where students learn multiple subjects or movements at once, rather than cramming one subject for a long period of time. The paper begins with an explanation of five stages of neurodevelopment: neurogenesis, encoding, consolidation, reconsolidation, myelination. Then, I explain the three practices and how they work within the stages of neurodevelopment. I conclude each section with examples of how tango teachers are currently implementing these practices, along with suggestions for further integration.

Traumatic brain injury (TBI) refers to brain damage resulting from an external force resulting in temporary or permanent impairment of cognitive, physical, and psychosocial functions. Following TBI, widespread neuronal loss occurs, and ischemic and inflammatory processes can greatly increase the extent of neural injury beyond the initial mechanical injury. Microglia are specialized immune cells in the brain that constantly surveil the extracellular environment and respond rapidly to damage by proliferating, phagocytosing debris, and releasing cytokines and chemokines that orchestrate recruitment and regulation of peripheral immune cells to the injured brain post-TBI. With the emergence of chemogenetics, a method by which engineered proteins interact with previously unrecognized chemical activators, inhibitory control can be exerted over microglia activation in a highly specific fashion allowing for precise targeting of brain regions and fewer off-target effects relative to traditional pharmacological approaches. The Weinstein lab aims to examine the effects of targeted inhibition of microglia activation using G-protein coupled receptors called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Normally, following traumatic brain injury, the CD68 promoter region is upregulated, resulting in increased microglia expression. However, the inserted HM4Di DREADD gene utilizes this promoter to express the DREADD receptor, and the downstream effects result in neural inflammatory response inhibition. We hypothesize that microglial inhibition will reduce proliferation and local cytokine levels after TBI, thus modulating the inflammatory microenvironment, especially when inhibition is initiated early after TBI. To determine efficacy of DREADDs, we quantify microglia number and proliferation using immunohistochemistry and stereology. We use computer software to capture multi-channel fluorescent images and montages for use in cell counting following stereological methods for random, unbiased sampling of cortical tissue across the TBI epicenter and penumbra. We anticipate that regions expressing activated DREADDs, which should inhibit microglial activation, will have reduced microglia post-TBI relative to controls.

Mitochondrial disease refers to a group of disorders that affects the mitochondria and therefore influences energy production and metabolism. The main purpose of this study is to determine the impact of pharmacological interventions with known age-delaying activity on neurological mitochondrial disease. In order to achieve this, a mouse model of mitochondrial disease lacking a subunit of the NADH-Ubiquinone Oxidoreductase Complex (Ndufs4-/-) was used to conduct experiments. This model recapitulates Leigh Syndrome, a childhood mitochondrial disease characterized by progressive loss of psychomotor activity, retarded growth, and death within the first three years of life. Inhibition of mTOR (mechanistic Target of Rapamycin) with rapamycin increases lifespan across multiple model organisms. Rapamycin also increases lifespan in Ndufs4-/- mice. In this study, we tested whether acarbose, another drug that extends lifespan in mice, could also extend lifespan in Ndufs4-/- mice. Mice treated with acarbose had longer lifespan compared to untreated animals, and a significant delay in the onset of neurological symptoms. We also obtained brain tissue from these mice to determine whether rapamycin and acarbose are acting on the same biochemical pathways to rescue disease in these animals. Western blot analysis of brain protein extract from rapamycin treated mice showed no phosphorylation of S6 ribosomal protein, a marker of mTOR activity. Conversely, mice treated with acarbose showed phosphorylation of S6 ribosomal protein in the brain, suggesting that acarbose does not inhibit mTOR. Although both drugs prolonged lifespan in this model, these results suggest that they do not act on the same biochemical mechanisms. However, both rapamycin and acarbose appear to restore the NAD+/NADH ratio, reduce accumulation of glycolytic intermediates, and reduce acetylation of mitochondrial proteins in the brains of Ndufs4-/- mice, suggesting that the two drugs may have convergent effects on disease suppression.

Protein homeostasis is an essential cellular process that directs cellular pathways involved in maintaining the integrity of the proteome. An important part of maintaining protein homeostasis is the degradation of misfolded and damaged proteins. This degradation primarily occurs by two major pathways: autophagy and proteasome. The proteasome is a protein complex that degrades unneeded and damaged proteins by proteolysis. The proteasome system consists of the Ubiquitin-Dependent Proteasome System (UPS) and the Ubiquitin Independent Proteasome System (UIPS). Previous studies suggest that the UIPS, which consists of the 20S Core Particle (CP) preferentially degrades proteins that accumulate with age and in age-related neurodegenerative diseases such as Alzheimer’s (AD) and Huntington’s (HD). Proteasome Activator (PA) drugs stimulate the 20S CP, and recent evidence suggests that these therapies can lead to the preferential degradation of misfolded proteins in vitro. The goal of our project was to characterize the effects of PA drugs in vivo using C. elegans animal models of AD and HD. These transgenic models express human amyloid-beta and huntingtin proteins, which we quantified using Western Blotting after treatment with the drugs. We hypothesized that PA drugs would reduce the amount of amyloid-beta and huntingtin proteins and lead to an overall decrease in insoluble proteins that accumulate with age in both the wildtype and disease backgrounds. Our preliminary data suggest that some PA drugs improve survival in a wildtype background, as well as in a neurodegenerative model of AD. If these drugs are also effective in reducing toxic protein aggregates, they would represent an exciting avenue for determining the therapeutic potential of small molecule stimulators for the treatment of protein aggregation diseases.