P4‐154: The Protein Quality Control Protein, Ubiquilin‐2, Regulates Tau Accumulation

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152960/1/alzjjalz2019063816.pd

Indian Ocean humpback dolphin (Sousa plumbea) movement patterns along the South African coast

1. The Indian Ocean humpback dolphin was recently uplisted to ‘Endangered’ in the recent South African National Red List assessment. Abundance estimates are available from a number of localized study sites, but knowledge of movement patterns and population linkage between these sites is poor. A national research collaboration, the SouSA project, was established in 2016 to address this key knowledge gap. Twenty identification catalogues collected between 2000 and 2016 in 13 different locations were collated and compared. 2. Photographs of 526 humpback dolphins (all catalogues and photos) were reduced to 337 individuals from 12 locations after data selection. Of these, 90 matches were found for 61 individuals over multiple sites, resulting in 247 uniquely, well‐marked humpback dolphins identified in South Africa. 3. Movements were observed along most of the coastline studied. Ranging distances had a median value of 120 km and varied from 30 km up to 500 km. Long‐term site fidelity was also evident in the data. Dolphins ranging along the south coast of South Africa seem to form one single population at the western end of the species' global range. 4. Current available photo‐identification data suggested national abundance may be well below previous estimates of 1000 individuals, with numbers possibly closer to 500. Bearing in mind the poor conservation status of the species in the country, the development of a national Biodiversity Management Plan aimed at ensuring the long‐term survival of the species in South Africa is strongly recommended. At the same time, increased research efforts are essential, particularly to allow for an in‐depth assessment of population numbers and drivers of changes therein. 5. The present study clearly indicates the importance of scientific collaboration when investigating highly mobile and endangered species.This collaborative research project was funded by the South African Network for Coastal and Oceanic Research (SANCOR), the National Research Foundation (NRF), and the University of Pretoria.http://wileyonlinelibrary.com/journal/aqc2019-02-01hj2018Mammal Research Institut

Building Connections between University of Michigan Scientists and Michigan's Native American Communities

Native Americans are currently underrepresented in science and in higher education. Taken together with the fact that indigenous viewpoints are frequently diminished in the United States, we have a severe lack of representation of the knowledge and perspectives of Native Americans, especially in the sciences. We aim to form collaborations with Michigan Native American tribes that will serve three main goals: 1. Bring awareness of different fields of science and opportunities to meet scientists to Native American youth. 2. Bring knowledge and awareness of dementia and aging to Native American elders. 3. Forge connections to the Native American community to learn about concerns related to science, medicine and higher education to inform research and education efforts at the University of Michigan. It is clear that increasing representation of Native Americans in science will lead to better scientific discoveries. Innovative and creative ideas are not made when everyone thinks in the same way.Program in Biomedical SciencesCentral Student GovernmentGinsberg CenterSACNAShttps://deepblue.lib.umich.edu/bitstream/2027.42/148851/1/Pistorius.ppt

UBQLN2 Function and Dysfunction in the Central Nervous System and Synucleinopathies

Protein accumulation and aggregation are hallmarks of many neurodegenerative disorders. The protein alpha-synuclein (a-syn) accumulates in Parkinson’s disease and several other age-related diseases, collectively referred to as the synucleinopathies. When phosphorylated at serine 129 (pS129), a-syn aggregates and ultimately forms Lewy bodies in neurons. A better understanding of how a-syn is regulated both normally and in disease would provide insight into the development and progression of Parkinson’s disease pathology. The protein quality control factor, Ubiquilin-2 (UBQLN2) has been implicated in multiple neurodegenerative diseases including in Parkinson’s disease and other synucleinopathies. However, it is not known whether UBQLN2 regulates a-syn. In this dissertation, I aim to determine if UBQLN2 regulates a-syn in normal and disease states and to elucidate the mechanism by which UBQLN2 acts. UBQLN2 is associated with synucleinopathies through its presence in Lewy bodies. However, it is unknown whether UBQLN2 is simply sequestered in these inclusions or whether its presence in Lewy bodies reflects a function of the protein in handling a-syn. In chapter two, I use cellular and mouse models to show that UBQLN2 regulates a-syn levels, particularly pS129, and demonstrate that UBQLN2 knock-out leads to total and pS129 a-syn accumulation. Pharmacological inhibition of the proteasome revealed that UBQLN2 targets pS129 for proteasomal degradation. Moreover, in brain tissue from PD and transgenic mice expressing pathogenic a-syn (A53T), endogenous UBQLN2 becomes more insoluble. Collectively, these studies support a previously unknown role for UBQLN2 in directly regulating pathological forms of a-syn and indicate that UBQLN2 dysregulation in disease may contribute to a-syn mediated toxicity. In chapter three I describe the unexpected finding that UBQLN2 overexpression in UBQLN2 transgenic mice leads to severe retinal degeneration in a dose-dependent manner. Immunofluorescence of retinas from UBQLN2 transgenic mice overexpressing WT or a pathogenic form of UBQLN2 (P506T) show punctate UBQLN2 expression in the outer retina, whereas endogenous UBQLN2 expression is diffuse and largely contained to the inner retina. I used optical coherence tomography to show that retinal degeneration in mice begins early and is rapid; with high UBQLN2 expression, degeneration began as early as four weeks of age, with a total loss of outer retinal layers by eight weeks of age. Retinal degeneration was less profound in P506T mice; retinas began thinning after six months of age, leading to a robust loss of outer retina cells by nine months of age. Disruption of proteostasis has been linked to multiple retinopathies, and our studies suggest that dysregulation of the protein quality control factor UBQLN2 is deleterious to retinal health. UBQLN2 knock-out did not robustly affect retinal thickness but may negatively impact retinal health at one year of age, suggesting that UBQLN2 is not necessary for proteostasis in the retina. UBQLN2 dysregulation in WT overexpressing and P506T retinas may lead to dysfunctional protein degradation, perhaps through sequestration of key protein quality control factors. These studies introduce the retina as a robust model for studies investigating UBQLN2 function and dysfunction and may have implications for retinal health in neurodegeneration. Chapter four outlines ongoing studies and future directions to further elucidate UBQLN2 function. This dissertation reveals a novel role for UBQLN2 as a regulator of pathogenic a-syn in disease and establishes the retina as a model for future studies of UBQLN2 function and dysfunction in protein quality control pathways.PHDNeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/172747/1/sspisto_1.pd

Advances in Deep Brain Stimulation: From Mechanisms to Applications.

Deep brain stimulation (DBS) is an effective therapy for various neurologic and neuropsychiatric disorders, involving chronic implantation of electrodes into target brain regions for electrical stimulation delivery. Despite its safety and efficacy, DBS remains an underutilized therapy. Advances in the field of DBS, including in technology, mechanistic understanding, and applications have the potential to expand access and use of DBS, while also improving clinical outcomes. Developments in DBS technology, such as MRI compatibility and bidirectional DBS systems capable of sensing neural activity while providing therapeutic stimulation, have enabled advances in our understanding of DBS mechanisms and its application. In this review, we summarize recent work exploring DBS modulation of target networks. We also cover current work focusing on improved programming and the development of novel stimulation paradigms that go beyond current standards of DBS, many of which are enabled by sensing-enabled DBS systems and have the potential to expand access to DBS

UBQLN2 regulation of α- synuclein in synucleinopathies

BackgroundThe protein quality control protein ubiquilin- 2 (UBQLN2) is implicated in synucleinopathies due to its accumulation in Lewy body diseases. However, little is known about how it may interact with and clear α- synuclein (α- syn). This study aimed to define the role of UBQLN2 in handling α- syn.MethodTo evaluate whether UBQLN2 regulates α- syn, we measured levels of α- synuclein in HEK- 293 cells transiently expressing or deleted of UBQLN2. To evaluate whether UBQLN2 regulates α- syn clearance in the nervous system in vivo, we used western blot to measure total α- syn or phosphorylated human α- syn (pS129) levels in multiple transgenic mouse lines including: UBQLN2 overexpressing mice (Ub2- hi), UBQLN2 knock- out mice (Ub2- KO), and A53T α- syn mice crossed to either Ub2- hi or Ub2- KO mice. To assess changes in UBQLN2 solubility in synucleinopathies we measured levels of UBQLN2 by Western blot in PBS- soluble versus sarkosyl- soluble brain lysates from PD and LBD human brains and from A53T mouse brains.ResultIn vitro, UBQLN2 significantly decreased levels of soluble α- syn. In vivo, endogenous insoluble α- syn levels are decreased in Ub2- hi mice, while total endogenous α- syn levels are significantly increased in Ub2- KO mice. Total α- syn and pS129 levels were unchanged in A53TxUb2- hi mice versus A53T controls, but were significantly increased in A53TxUb2- KO mice. Solubility studies revealed increased insoluble UBQLN2 levels in human LBD and mouse A53T brains.ConclusionWhile UBQLN2 is known to colocalize with a- syn in disease, our results support a functional role for UBQLN2 in regulating α- syn levels. Further, we show that UBQLN2 solubility is altered in synucleinopathies. In disease, a change in UBQLN2 solubility may indicate a loss of its ability to handle a- syn, contributing to a- syn toxicity. Ongoing studies seek to elucidate the mechanism by which UBQLN2 handles, and potentially clears, α- syn.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/171612/1/alz055354.pd

Ubiquilin‐2 exacerbates tau toxicity in vivo

BackgroundUbiquilin‐2 (UBQLN2) is a protein quality control protein involved primarily in shuttling ubiquitinated substrates to the proteasome for degradation and by modulating autophagy. UBQLN2 has been implicated in neurodegenerative disease due to its accumulation in neuropathological deposits and its potential role in regulating protein dyshomeostasis common across different neurodegenerative disorders. The relationship of UBQLN2 to one of the most common aggregating proteins in disease, tau, is unknown.MethodTo evaluate whether UBQLN2 regulates tau clearance, we assessed levels of tau in human embryonic kidney‐293 cells with and without UBQLN2. To determine whether UBQLN2 acts on tau in vivo, P301S tau transgenic mice were crossed with UBQLN2 transgenic and knockout mice and brain levels of tau were assessed at 3, 6 and 9 months of age. To define changes in UBQLN2 in human disease, we measured levels of soluble and insoluble UBQLN2 in human tauopathy brain tissue.ResultCo‐expressed UBQLN2 markedly lowered levels of tau in a cellular model. Conversely, siRNA knockdown of UBQLN2 significantly elevated levels of tau. Surprisingly, a UBQLN2 mutant incapable of binding ubiquitin was more effective at lowering tau than wildtype UBQLN2, suggesting that ubiquitin‐independent pathways may allow UBQLN2 to “handle” tau. In contrast, wildtype UBQLN2 overexpression in vivo did not alter total levels of tau at 3, 6 or 9 months of age. However, UBQLN2 overexpression specifically increased phosphorylated tau while UBQLN2 knockout decreased phosphorylated tau at 9 months. Furthermore, UBQLN2 overexpression increased premature hindlimb paralysis and fatality. The possibility that UBQLN2 also undergoes alterations in disease was evidenced by the fact that UBQLN2 solubility is decreased in human brains with tau pathology.ConclusionOur findings highlight a new role for UBQLN2 in altering tau in the brain. Collectively, our results suggest that while on a rapid time scale UBQLN2 can decrease tau levels, long‐term expression of UBQLN2 in vivo exacerbates tau toxicity. Ongoing research will determine how changing UBQLN2 levels alters components of proteostasis pathways to affect tau toxicity and whether ubiquitin‐independent processes may compete with UBQLN2’s function as a ubiquitin‐proteasome shuttle factor to yield differential effects on tau toxicity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163894/1/alz046096.pd

Structural Insights Into Anthranilate Priming During Type II Polyketide Biosynthesis

The incorporation of non-acetate starter units during type II polyketide biosynthesis helps diversify natural products. Currently, there are few enzymatic strategies for the incorporation of non-acetate starter units in type II PKS pathways. Here we report the crystal structure of AuaEII, the anthranilate:CoA ligase responsible for the generation of anthranoyl-CoA, which is used as a starter unit by a type II PKS during aurachin biosynthesis. We present structural and protein sequence comparisons to other aryl:CoA ligases. We also compare the AuaEII crystal structure to a model of a CoA ligase homologue, AuaE, which is present in the same gene cluster. AuaE is predicted to have the same fold as AuaEII, but instead of CoA ligation, AuaE catalyzes acyl transfer of anthranilate from anthranoyl-CoA to the acyl carrier protein (ACP). Together, this work provides insight into the molecular basis for starter unit selection of anthranilate during type II PKS biosynthesis

Ventral Tegmental Area GABA Neurons Are Resistant to GABA(A) Receptor-Mediated Inhibition During Ethanol Withdrawal

The neural mechanisms underlying alcohol dependence are not well-understood. GABAergic neurons in the ventral tegmental area (VTA) are a relevant target for ethanol. They are inhibited by ethanol at physiologically-relevant levels in vivo and display marked hyperexcitability during withdrawal. In the present study, we examined the effects of the GABA(A) receptor agonist muscimol on VTA neurons ex vivo following withdrawal from acute and chronic ethanol exposure. We used standard cell-attached mode electrophysiology in the slice preparation to evaluate the effects of muscimol on VTA GABA neuron firing rate following exposure to acute and chronic ethanol in male CD-1 GAD-67 GFP mice. In the acute condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after a single in vivo dose of saline or ethanol. In the chronic condition, the effect of muscimol on VTA neurons was evaluated 24 h and 7 days after either 2 weeks of twice-daily IP ethanol or saline or following exposure to chronic intermittent ethanol (CIE) vapor or air for 3 weeks. VTA GABA neuron firing rate was more sensitive to muscimol than DA neuron firing rate. VTA GABA neurons, but not DA neurons, were resistant to the inhibitory effects of muscimol recorded 24 h after a single ethanol injection or chronic ethanol exposure. Administration of the NMDA receptor antagonist MK-801 before ethanol injection restored the sensitivity of VTA GABA neurons to muscimol inhibition. Seven days after ethanol exposure, VTA GABA neuron firing rate was again susceptible to muscimol's inhibitory effects in the acute condition, but the resistance persisted in the chronic condition. These findings suggest that VTA GABA neurons exclusively undergo a shift in GABA(A) receptor function following acute and chronic exposure. There appears to be transient GABA(A) receptor-mediated plasticity after a single exposure to ethanol that is mediated by NMDA glutamate receptors. In addition, the resistance to muscimol inhibition in VTA GABA neurons persists in the dependent condition, which may contribute to the the hyperexcitability of VTA GABA neurons and inhibition of VTA DA neurons during withdrawal as well as the motivation to seek alcohol

Structural Insights into Anthranilate Priming during Type II Polyketide Biosynthesis

The incorporation of nonacetate starter units during type II polyketide biosynthesis helps diversify natural products. Currently, there are few enzymatic strategies for the incorporation of nonacetate starter units in type II polyketide synthase (PKS) pathways. Here we report the crystal structure of AuaEII, the anthranilate:CoA ligase responsible for the generation of anthraniloyl-CoA, which is used as a starter unit by a type II PKS in aurachin biosynthesis. We present structural and protein sequence comparisons to other aryl:CoA ligases. We also compare the AuaEII crystal structure to a model of a CoA ligase homologue, AuaE, which is present in the same gene cluster. AuaE is predicted to have the same fold as AuaEII, but instead of CoA ligation, AuaE catalyzes acyl transfer of anthranilate from anthraniloyl-CoA to the acyl carrier protein (ACP). Together, this work provides insight into the molecular basis for starter unit selection of anthranilate in type II PKS biosynthesis