applying-ratwalker-system-for-gait-analysis-genetic-rat-model

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

University of Nebraska Medical Center

Human immunodeficiency virus (HIV) accesses the brain early in infection and can lead to neurocognitive disorders. The brain can also serve as a viral reservoir, but how virus is controlled in the brain is unknown. To examine this, CD8-depleting monoclonal antibody was injected into the cerebrospinal fluid of rhesus monkeys with chronic simian immunodeficiency virus (SIV) infection. This treatment led to the rapid increase of SIV in the brain. Virus in the brain is maintained by active suppression from the host immune system. This dynamic interaction can be manipulated in efforts to control and eradicate virus from the brain and other reservoirs. 

CD8+ T cells maintain suppression of simian immunodeficiency virus in the central nervous system.

Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders. 

MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease.

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell-cell communication is critical. One relatively unexplored form of cell-cell communication in TBI is extracellular vesicles (EVs). These membrane-bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV-associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR-212 decreased, while miR-21, miR-146, miR-7a, and miR-7b were significantly increased with injury, with miR-21 showing the largest change between conditions. The expression of miR-21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR-21-expressing neurons were activated microglia. The concurrent increase in miR-21 in EVs with the elevation of miR-21 in neurons, suggests that miR-21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell-cell communication not previously described in TBI.

Traumatic brain injury increases levels of miR-21 in extracellular vesicles: implications for neuroinflammation.

Traumatic brain injury (TBI) produces profound and lasting neuroinflammation that has both beneficial and detrimental effects. Recent evidence has implicated microRNAs (miRNAs) in the regulation of inflammation both in the periphery and the CNS. We examined the expression of inflammation associated miRNAs in the context of TBI using a mouse controlled cortical impact (CCI) model and found increased levels of miR-21, miR-223 and miR-155 in the hippocampus after CCI. The expression of miR-155 was elevated 9-fold after CCI, an increase confirmed by  hybridization (ISH). Interestingly, expression of miR-155 was largely found in neuronal nuclei as evidenced by co-localization with DAPI in MAP2 positive neurons. In miR-155 knock out (KO) mice expression of type I interferons  and , as well as IFN regulatory factor 1 and IFN-induced chemokine  was decreased after TBI relative to wild type (WT) mice. Unexpectedly, miR-155 KO mice had increased levels of microglial marker Iba1 and increased neuronal degeneration as measured by fluoro-jade C (FJC) staining, suggesting a neuroprotective role for miR-155 in the context of TBI. This work demonstrates a role for miR-155 in regulation of the IFN response and neurodegeneration in the aftermath of TBI. While the presence of neuronal nuclear miRNAs has been described previously, their importance in disease states is relatively unknown. Here, we show evidence of dynamic regulation and pathological function of a nuclear miRNA in TBI.

Induction of miR-155 after Brain Injury Promotes Type 1 Interferon and has a Neuroprotective Effect.

The use of antiretroviral (ARV) drugs with central nervous system (CNS) penetration effectiveness (CPE) may be useful in the treatment of HIV-associated neurocognitive disorder (HAND) as well as targeting a CNS reservoir in strategies to achieve a functional cure for HIV. However, increased cognitive deficits are linked to at least one of these drugs (efavirenz). As mitochondrial dysfunction has been found with a number of ARVs, and as such can affect neuronal function, the objective of this study was to assess the effects of ARV with high CPE for toxicological profiles on presynaptic nerve terminal energy metabolism. This subcellular region is especially vulnerable in that a constant supply of ATP is required for the proper maintenance of neurotransmitter release and uptake supporting proper neuronal function. We evaluated the effects of acute treatment with ten different high CPE ARVs from five different drug classes on rat cortical and striatal nerve terminal bioenergetic function. While cortical nerve terminal bioenergetics were not altered, striatal nerve terminals exposed to efavirenz, nevirapine, abacavir, emtricitabine, zidovudine, darunavir, lopinavir, raltegravir, or maraviroc (but not indinavir) exhibit reduced mitochondrial spare respiratory capacity (SRC). Further examination of efavirenz and maraviroc revealed a concentration-dependent impairment of striatal nerve terminal maximal mitochondrial respiration and SRC as well as a reduction of intraterminal ATP levels. Depletion of ATP at the synapse may underlie its dysfunction and contribute to neuronal dysfunction in treated HIV infection.

Central nervous system-penetrating antiretrovirals impair energetic reserve in striatal nerve terminals.

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.

Creation of a long-acting rilpivirine prodrug nanoformulation.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.

A year-long extended release nanoformulated cabotegravir prodrug.

DJ-1 is a multifunctional protein affecting different biological and cellular processes. In addition, DJ-1 has roles in regulating mitochondrial function. Loss-of-function mutations in DJ-1 were found to cause an autosomal recessive form of Parkinson's disease. One of the main pathological features of PD is loss of dopamine neurons in the nigrostriatal pathway. DJ-1 knockout (KO) rats exhibit progressive nigral neurodegeneration with about 50% dopaminergic cell loss at 8 months of age. In order to assess the effects of DJ-1 deficiency on neuronal mitochondria prior to neuron loss, we performed proteomic analysis of synaptic mitochondria isolated from the striatum, the location of nigrostriatal pathway nerve terminals, of 3-month-old DJ-1 KO rats. In total, 371 mitochondrial proteins were quantified, and of these 76 were differentially expressed in DJ-1 KO rats. Proteins perturbed by the loss of DJ-1 were involved in several mitochondrial functional pathways, including the tricarboxylic acid cycle and electron transport chain. Thus, synaptic mitochondrial respiration was measured and showed a significant change due to DJ-1 deficiency. The dataset generated here highlights the role of synaptic mitochondria in PD associated with DJ-1. This study improves our understanding of DJ-1 effects in a complex tissue environment and the synaptic mitochondrial changes that accompany its loss.

Deletion of DJ-1 in rats affects protein abundance and mitochondrial function at the synapse.

Both substance use disorder and HIV infection continue to affect many individuals. Both have untoward effects on the brain, and the two conditions often co-exist. In the brain, macrophages and microglia are infectable by HIV, and these cells are also targets for the effects of drugs of abuse, such as the psychostimulant methamphetamine. To determine the interaction of HIV and methamphetamine, we isolated microglia and brain macrophages from SIV-infected rhesus monkeys that were treated with or without methamphetamine. Cells were subjected to single-cell RNA sequencing and results were analyzed by statistical and bioinformatic analysis. In the animals treated with methamphetamine, a significantly increased proportion of the microglia and/or macrophages were infected by SIV. In addition, gene encoding functions in cell death pathways were increased, and the brain-derived neurotropic factor pathway was inhibited. The gene expression patterns in infected cells did not cluster separately from uninfected cells, but clusters comprised of microglia and/or macrophages from methamphetamine-treated animals differed in neuroinflammatory and metabolic pathways from those comprised of cells from untreated animals. Methamphetamine increases CNS infection by SIV and has adverse effects on both infected and uninfected microglia and brain macrophages, highlighting the dual and interacting harms of HIV infection and drug abuse on the brain.

Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis.

Article	Year
CD8+ T cells maintain suppression of simian immunodeficiency virus in the central nervous system. The Journal of infectious diseases\| PubMed ID: 25035516	2015
MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease. PLoS pathogens\| PubMed ID: 26154133	2015
Traumatic brain injury increases levels of miR-21 in extracellular vesicles: implications for neuroinflammation. FEBS open bio\| PubMed ID: 27516962	2016
Induction of miR-155 after Brain Injury Promotes Type 1 Interferon and has a Neuroprotective Effect. Frontiers in molecular neuroscience\| PubMed ID: 28804446	2017
Central nervous system-penetrating antiretrovirals impair energetic reserve in striatal nerve terminals. Journal of neurovirology\| PubMed ID: 28895059	2017
Creation of a long-acting rilpivirine prodrug nanoformulation. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 31491432	2019
A year-long extended release nanoformulated cabotegravir prodrug. Nature materials\| PubMed ID: 32341511	2020
Deletion of DJ-1 in rats affects protein abundance and mitochondrial function at the synapse. Scientific reports\| PubMed ID: 32792613	2020
Methamphetamine Increases the Proportion of SIV-Infected Microglia/Macrophages, Alters Metabolic Pathways, and Elevates Cell Death Pathways: A Single-Cell Analysis. Viruses\| PubMed ID: 33198269	2020

Benjamin G. Lamberty

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