Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. Our lab previously engineered a genetically-encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of co-cultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric redox imaging in Drosophila retinas. The proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms. In addition, we have used these tools that monitor cellular redox, to study oxidative stress and ROS changes in Parkinson’s disease models. Here, we have established cellular models for studying Parkinson’s disease causing LRRK2 mutations to create a platform for future work to explore the relationship between PD associated LRRK2 variants and oxidative stress.

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In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

BACKGROUND: Neuronal remodeling in human heart disease is not well understood. METHODS: Stellate ganglia from patients with cardiomyopathy (CMY) and refractory ventricular arrhythmias undergoing cardiac sympathetic denervation (n = 8), and from organ donors with normal hearts (n = 8) collected at the time of organ procurement were compared. Clinical data on all subjects were reviewed. Electron microscopy (EM), histologic, and immunohistochemical assessments of neurotransmitter profiles, glial activation and distribution, and lipofuscin deposition, a marker of oxidative stress, were quantified. RESULTS: In CMY specimens, lipofuscin deposits were larger, and present in more neurons (26.3% ± 6.3% vs. 16.7% ± 7.6%, P < 0.043), than age-matched controls. EM analysis revealed extensive mitochondrial degeneration in CMY specimens. T cell (CD3+) infiltration was identified in 60% of the CMY samples, with one case having large inflammatory nodules, while none were identified in controls. Myeloperoxidase-immunoreactive neutrophils were also identified at parenchymal sites distinct from inflammatory foci in CMY ganglia, but not in controls. The adrenergic phenotype of pathologic samples revealed a decrease in tyrosine hydroxylase staining intensity compared with controls. Evaluation of cholinergic phenotype by staining for the vesicular acetylcholine transporter revealed a low but comparable number of cholinergic neurons in ganglia from both groups and demonstrated that preganglionic cholinergic innervation was maintained in CMY ganglia. S100 staining (a glial cell marker) demonstrated no differences in glial distribution and relationship to neurons; however, glial activation demonstrated by glial fibrillary acidic protein (GFAP) staining was substantially increased in pathologic specimens compared with controls. CONCLUSIONS: Stellate ganglia from patients with CMY and arrhythmias demonstrate inflammation, neurochemical remodeling, oxidative stress, and satellite glial cell activation. These changes likely contribute to excessive and dysfunctional efferent sympathetic tone, and provide a rationale for sympathectomy as a treatment for arrhythmias in this population. FUNDING: This work was made possible by support from NIH grants HL125730 to OAA, GM107949 to DBH, and HL084261 and OT2OD023848 to KS.

Sociality shapes patterns of senescence, evidenced by the remarkable lifespan plasticity of social insect queens and workers. Ants, exemplars of eusociality, provide diverse systems to explore the sociobiology of senescence by examining how sterile workers partition colony labor over their lifespans, and how neurobiological factors affect transitions among social roles and age-related task performance efficacies. Integrating sociobiology, senescence theory, and neurobiology, I examined the relationship of chronological age and social behavior during the ~140-day lifespan of workers of the ant Pheidole dentata. I critically analyzed programmed senescence in respect to the sociobiology of worker longevity and evaluated how large colony size achieved through selection for extended worker lifespan enhances colony fitness. My study found no support for worker programmed senescence. Further testing senescence theory, I determined if workers declined behaviorally as they aged due to increased apoptotic cell death and changes in synaptic complexes associated with higher-order processing in the brain. Using robust behavioral assays I found aging was not correlated with declines in sensory responsiveness or motor functions associated with foraging, nursing, and prey-capture tasks, or activity level and phototaxis. Old minor workers (95 days) followed pheromone trails for greater distances than 20-day old minors and showed higher activity levels, suggesting improvement in behavioral performance. Neural substrates likely underscoring task performance were maintained with age: synaptic complex density was constant and apoptosis was unchanged with age. Sensory and motor control brain regions did not show age-related increases in neurodegeneration. Worker spatial location predicted social role independent of age: foragers exhibited higher activity levels and more aggressive predatory behavior than nurses. Serotonin and dopamine titers increased from 20 to 120 days but showed no clear correlation with social role. Pharmacological manipulations of brain serotonin had no effect on brood care, predatory response, activity, or phototaxis. Finally, I assessed arborization of a serotonergic neuron hypothesized to underscore task performance to determine how aging across subcastes influences neuronal structure. Major workers showed greater branching complexity than minors and an age-related increase in arbor complexity. P. dentata workers appear to show negligible behavioral and neural senescence throughout their lifespans.

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New models in which aging-related neurodegeneration more closely resembling the combination of pathologies that develop in aging humans, are needed. The fish Nothobranchius, which naturally develops such pathologies over the course of its short lifespan, is one such model. This review compares the lifespans and pathologies of different Nothobranchius strains to those of current vertebrate models of aging. Furthermore, existing data pertaining to neurodegeneration in these fish is discussed in the context of their reported neuropathologies, along with open questions related to mammalian chronopathologies. Specifically, the evidence for a Parkinson’s disease-like pathology is discussed. Neurogenesis and age-related changes therein are discussed in the context of siRNA and neurodegeneration. We also discuss changes in the expression of neuropeptide Y in relation to the brain-gut axis and how these change with age. Age-related behavioral changes are discussed, along with the assays used in their evaluation. Genetic discoveries are outlined and discussed with a view on DJ-1/NRF2 signaling in N. furzeri, and insights gained from comparative genomics and siRNA studies. Finally, research focus areas are highlighted, and a case is made for the utility of these fish in the study of aging-related neurodegeneration, and to screen for environmental risk factors of aging-related neuropathology.

Metabotropic glutamate receptor 5 (mGluR5) is a key regulator of numerous brain functions including memory, cognition, and motor behavior. Dysregulation of mGluR5 signaling is evident in Huntington's disease (HD) neuropathology, an inherited, neurodegenerative disease characterized with progressive deterioration in motor, cognitive, and psychiatric functions. In this context, two cellular proteins draw particular interest for this thesis: vesicular glutamate transporter 3 (VGLUT3) and optineurin (OPTN). VGLUT3 modulates glutamate release from selected neurons that are affected by HD, while OPTN is a mGluR5-interacting protein and contributes to neuronal vulnerability in HD. However, current evidence on their involvement in mGluR5 signaling and HD pathogenesis is still lacking. Using VGLUT3 knockout (VGLUT3⁻ᐟ⁻) mice, we showed that this transporter dynamically regulated glutamate receptor densities in different brain regions. Of note, VGLUT3 deletion upregulated mGluR5 in the cerebral cortex and the striatum, unlike the hippocampus which exhibited reduced mGluR5 cell surface densities. We then crossed VGLUT3⁻ᐟ⁻ mice with the zQ175 knock-in mouse model of HD (zQ175:VGLUT3⁻ᐟ⁻) to assess the impact of VGLUT3 transmission loss on HD progression. The longitudinal behavioral assessment revealed that VGLUT3 ablation rescued the deficits in motor coordination and short-term memory in both male and female zQ175 mice throughout 15 months of age. Furthermore, VGLUT3 deletion rescued striatal cell loss likely via activation of Akt and ERK1/2 cellular pathways, with no impact on total mutant huntingtin aggregation or the associated microgliosis. To delineate the role of OPTN in mGluR5 signaling, we employed a CRISPR/Cas9 OPTN-deficient cell line and global OPTN knockout mice. We demonstrated that OPTN was essential for mGluR5-mediated canonical signaling and ERK1/2 activation in both the striatal cell line, STHdh^Q7/Q7, and acute hippocampal slices. We then showed that OPTN deletion impaired autophagic machinery via GSK3β/ZBTB16 and mTOR/ULK1 signaling pathways downstream of mGluR5. This work offers novel insights into the molecular roles of VGLUT3 transmission and OPTN in mGluR5 signaling and provides a rationale for their targeting to therapeutically mitigate pathological mGluR5 signaling in HD.

High doses of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") are known to reduce levels of various serotonergic markers outside of the raphe nuclei. To test the hypothesis that these deficits reflect a degeneration of distal axons/terminals, we investigated the effects of an MDMA binge (10mg/kg x 4) on the relative protein and genetic expression of several serotonergic markers in rats, as well as the effects of this compound on the quantity of serotonergic terminals in these animals. In experiment I, we examined whether MDMA alters serotonin transporter (SERT) levels as determined by lysate binding and immunoblotting analyses. Both methods of analysis revealed MDMA-induced reductions in regional SERT content. Experiment II investigated MDMA-induced changes in terminal-specific levels of SERT and the vesicular monoamine transporter 2 (VMAT-2) in the hippocampus, a region with sparse dopaminergic innervation, after lesioning noradrenergic input with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Animals were administered 100 mg/kg DSP-4 or saline 1 week prior to MDMA (or saline). As determined by immunoblotting of synaptosomal tissue, the DSP-4/MDMA group showed little change in hippocampal VMAT-2 protein expression compared to DSP-4/Saline controls, despite large reductions in SERT levels in all regions examined in the MDMA-treated animals. Experiment III examined whether MDMA alters genetic expression of SERT and VMAT-2. When compared to saline-treated controls, animals given MDMA showed a striking decrease in SERT gene expression (and a lesser effect on VMAT-2) in dorsal/median raphe as assessed by quantitative RT-PCR. Experiment IV(a) investigated the effects of MDMA on gene and protein expression of tryptophan hydroxylase (TPH) in the hippocampus. Levels of TPH protein were unchanged between treatment groups, while transcript levels were decreased 15-fold in the dorsal/median raphe. In experiment IV(b), flow cytometry was used to measure whether MDMA alters the quantity of serotonergic terminals in the hippocampus. MDMA-treated animals showed an increase in the number of serotonergic synaptosomes identified by co-labeling for synaptosome-associated protein of 25 kDa (SNAP-25) and TPH. These results demonstrate that MDMA causes substantial regulatory changes in the expression of serotonergic markers with no evidence for synaptic loss, questioning the need to invoke distal axotomy as an explanation of MDMA-related serotonergic deficits.