Soluble and insoluble lysates from the human A53T mutant α-synuclein transgenic mouse model induces α-synucleinopathy independent of injection site.

Pathological aggregation of a-synuclein (aS) is implicated in the pathogenesis of Parkinson's disease (PD) and other a-synucleinopathies. The current view is that neuron-to-neuron spreading of aS pathology contributes to the progression of a-synucleinopathy. We used an A53T mutant human aS transgenic mouse model (TgA53T) to examine whether the site of pathogenic aS inoculation affects the pattern of neuropathology and whether soluble and insoluble fractions derived from crude pathogenic tissue lysates exhibit differential capacities to initiate aS pathology. To test whether the inoculation site impacts the ultimate spatial/temporal patterns of aS pathology, aS preformed fibrils (PFF), or brain homogenates from TgA53T mice with a-synucleinopathy, were injected into the cortex/striatum, brain stem, or skeletal muscle. In all cases, inoculation of pathogenic aS induced end-stage motor dysfunction within ~100 days post-inoculation (dpi). Significantly, irrespective of the inoculation sites, ultimate distribution of the aS pathology was like that seen in normally aged TgA53T mice at end-stage, indicating that the intrinsic neuronal vulnerability is a significant determinant in the induction of aS pathology, even when initiated by inoculation of pathogenic aS. Temporal analysis of brain stem injected TgA53T mice show that initial aS pathology was seen by 30 days post-inoculation and inflammatory changes occur at later stages. To determine if the aS species with differential solubility are differentially pathogenic, brain lysates from end-stage TgA53Tmice were fractionated into highly soluble (S150) and insoluble (P150) fractions, as well as the endoplasmic reticulum (ER)-enriched fraction (P100). Significantly, all fractions were able to seed de novo aS pathology in vivo, when injected unilaterally into TgA53Tmice with the ER fractions being most pathogenic. Our results suggest that multiple aS species from brain can initiate the development of progressive aS pathology.

Loss of tau expression attenuates neurodegeneration associated with α-synucleinopathy.

BACKGROUND: Neuronal dysfunction and degeneration linked to α-synuclein (αS) pathology is thought to be responsible for the progressive nature of Parkinson's disease and related dementia with Lewy bodies. Studies have indicated bidirectional pathological relationships between αS pathology and tau abnormalities. We recently showed that A53T mutant human αS (HuαS) can cause post-synaptic and cognitive deficits that require microtubule-associated protein tau expression. However, the role of tau in the development of αS pathology and subsequent neuronal dysfunction has been controversial. Herein, we set out to determine the role of tau in the onset and progression of αS pathology (α-synucleinopathy) using a transgenic mouse model of α-synucleinopathy lacking mouse tau expression. METHODS: Transgenic mice expressing A53T mutant HuαS (TgA53T) were crossed with mTau-/- mice to generate TgA53T/mTau-/-. To achieve more uniform induction of α-synucleinopathy in mice, we used intramuscular injections of αS preformed fibrils (PFF) in non-transgenic (nTg), TgA53T, TgA53T/mTau-/-, and mTau-/- mice. Motor behavior was analyzed at 70 days post inoculation (dpi) of PFF and tissues for biochemical and neuropathological analysis were collected at 40 dpi, 70 dpi, and end stage. RESULTS: Loss of tau expression significantly delayed the onset of motor deficits in the TgA53T model and the progression of α-synucleinopathy disease, as evidenced by a significant reduction in histopathological and behavioral markers of neurodegeneration and disease, and a significant improvement in survival. In vitro application of PFF to primary mouse hippocampal neurons demonstrated no changes in PFF uptake and processing or pS129 αS aggregation as a function of tau expression. However, PFF-induced neurotoxicity, including morphological deficits in nTg neurons, was prevented with tau removal. CONCLUSIONS: Collectively, our data suggest that tau is likely acting downstream of αS pathology to affect neuronal homeostasis and survival. This work further supports the investigation of tau in α-synucleinopathies to identify novel disease-modifying therapeutic strategies.

UNC-45A Is Highly Expressed in the Proliferative Cells of the Mouse Genital Tract and in the Microtubule-Rich Areas of the Mouse Nervous System.

UNC-45A (Protein unc-45 homolog A) is a cytoskeletal-associated protein with a dual and non-mutually exclusive role as a regulator of the actomyosin system and a Microtubule (MT)-destabilizing protein, which is overexpressed in human cancers including in ovarian cancer patients resistant to the MT-stabilizing drug paclitaxel. Mapping of UNC-45A in the mouse upper genital tract and central nervous system reveals its enrichment not only in highly proliferating and prone to remodeling cells, but also in microtubule-rich areas, of the ovaries and the nervous system, respectively. In both apparatuses, UNC-45A is also abundantly expressed in the ciliated epithelium. As regulators of actomyosin contractility and MT stability are essential for the physiopathology of the female reproductive tract and of neuronal development, our findings suggest that UNC-45A may have a role in ovarian cancer initiation and development as well as in neurodegeneration.

Glycogen synthase kinase-3 inhibition rescues sex-dependent contextual fear memory deficit in human immunodeficiency virus-1 transgenic mice.

BACKGROUND AND PURPOSE: A significant number of HIV-1 patients on antiretroviral therapy develop HIV-associated neurocognitive disorders (HAND). Evidence indicate that biological sex may regulate HAND pathogenesis, but the mechanisms remain unknown. We investigated synaptic mechanisms associated with sex differences in HAND, using the HIV-1-transgenic 26 (Tg26) mouse model. EXPERIMENTAL APPROACH: Contextual- and cue-dependent memories of male and female Tg26 mice and littermate wild type mice were assessed in a fear conditioning paradigm. Hippocampal electrophysiology, immunohistochemistry, western blot, qRT-PCR and ELISA techniques were used to investigate cellular, synaptic and molecular impairments. KEY RESULTS: Cue-dependent memory was unaltered in male and female Tg26 mice, when compared to wild type mice. Male, but not female, Tg26 mice showed deficits in contextual fear memory. Consistently, only male Tg26 mice showed depressed hippocampal basal synaptic transmission and impaired LTP induction in area CA1. These deficits in male Tg26 mice were independent of hippocampal neuronal loss and microglial activation but were associated with increased HIV-1 long terminal repeat mRNA expression, reduced hippocampal synapsin-1 protein, reduced BDNF mRNA and protein, reduced AMPA glutamate receptor (GluA1) phosphorylation levels and increased glycogen synthase kinase 3 (GSK3) activity. Importantly, selective GSK3 inhibition using 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione increased levels of synapsin-1, BDNF and phosphorylated-GluA1 proteins, restored hippocampal basal synaptic transmission and LTP, and improved contextual fear memory in male Tg26 mice. CONCLUSION AND IMPLICATIONS: Sex-dependent impairments in contextual fear memory and synaptic plasticity in Tg26 mice are associated with increased GSK3 activity. This implicates GSK3 inhibition as a potential therapeutic strategy to improve cognition in HIV-1 patients.

α-Synucleinopathy associated c-Abl activation causes p53-dependent autophagy impairment.

BACKGROUND: Studies link c-Abl activation with the accumulation of pathogenic α-synuclein (αS) and neurodegeneration in Parkinson's disease (PD). Currently, c-Abl, a tyrosine kinase activated by cellular stress, is thought to promote αS pathology by either directly phosphorylating αS or by causing autophagy deficits. METHODS: αS overexpressing transgenic (Tg) mice were used in this study. A53T Tg mice that express high levels of human mutant A53TαS under the control of prion protein promoter. Two different approaches were used in this study. Natural aging and seeding model of synucleinopathy. In seeding model, intracortical/intrastriatal (IC/IS) stereotaxic injection of toxic lysates was done using tissue lysates from end-stage symptomatic mice. In this study, nilotinib and pifithrin-α was used as a c-Abl and p53 inhibitor, respectively. Both Tg and non-transgenic (nTg) mice from each group were subjected to nilotinib (10 mg/kg) or vehicle (DMSO) treatment. Frozen brain tissues from PD and control human cases were analyzed. In vitro cells study was implied for c-Abl/p53 genetic manipulation to uncover signal transduction. RESULTS: Herein, we show that the pathologic effects of c-Abl in PD also involve activation of p53, as c-Abl activation in a transgenic mouse model of α-synucleinopathy (TgA53T) and human PD cases are associated with the increased p53 activation. Significantly, active p53 in TgA53T neurons accumulates in the cytosol, which may lead to inhibition of autophagy. Thus, we hypothesized that c-Abl-dependent p53 activation contributes to autophagy impairment in α-synucleinopathy. In support of the hypothesis, we show that c-Abl activation is sufficient to inhibit autophagy in p53-dependent manner. Moreover, inhibition of either c-Abl, using nilotinib, or p53, using pifithrin-α, was sufficient to increase autophagic flux in neuronal cells by inducing phosphorylation of AMP-activated kinase (AMPK), ULK1 activation, and down-regulation of mTORC1 signaling. Finally, we show that pharmacological attenuation of c-Abl activity by nilotinib treatment in the TgA53T mouse model reduces activation of p53, stimulates autophagy, decreases accumulation αS pathology, and delays disease onset. CONCLUSION: Collectively, our data show that c-Abl activation by α-synucleinopathy causes p53 dependent autophagy deficits and both c-Abl and p53 represent therapeutic target for PD.

Tau is required for progressive synaptic and memory deficits in a transgenic mouse model of α-synucleinopathy.

Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically and neuropathologically highly related α-synucleinopathies that collectively constitute the second leading cause of neurodegenerative dementias. Genetic and neuropathological studies directly implicate α-synuclein (αS) abnormalities in PDD and DLB pathogenesis. However, it is currently unknown how αS abnormalities contribute to memory loss, particularly since forebrain neuronal loss in PDD and DLB is less severe than in Alzheimer's disease. Previously, we found that familial Parkinson's disease-linked human mutant A53T αS causes aberrant localization of the microtubule-associated protein tau to postsynaptic spines in neurons, leading to postsynaptic deficits. Thus, we directly tested if the synaptic and memory deficits in a mouse model of α-synucleinopathy (TgA53T) are mediated by tau. TgA53T mice exhibit progressive memory deficits associated with postsynaptic deficits in the absence of obvious neuropathological and neurodegenerative changes in the hippocampus. Significantly, removal of endogenous mouse tau expression in TgA53T mice (TgA53T/mTau-/-), achieved by mating TgA53T mice to mouse tau-knockout mice, completely ameliorates cognitive dysfunction and concurrent synaptic deficits without affecting αS expression or accumulation of selected toxic αS oligomers. Among the known tau-dependent effects, memory deficits in TgA53T mice were associated with hippocampal circuit remodeling linked to chronic network hyperexcitability. This remodeling was absent in TgA53T/mTau-/- mice, indicating that postsynaptic deficits, aberrant network hyperactivity, and memory deficits are mechanistically linked. Our results directly implicate tau as a mediator of specific human mutant A53T αS-mediated abnormalities related to deficits in hippocampal neurotransmission and suggest a mechanism for memory impairment that occurs as a consequence of synaptic dysfunction rather than synaptic or neuronal loss. We hypothesize that these initial synaptic deficits contribute to network hyperexcitability which, in turn, exacerbate cognitive dysfunction. Our results indicate that these synaptic changes present potential therapeutic targets for amelioration of memory deficits in α-synucleinopathies.

UNC-45A Is a Novel Microtubule-Associated Protein and Regulator of Paclitaxel Sensitivity in Ovarian Cancer Cells.

UNC-45A, a highly conserved member of the UCS (UNC45A/CRO1/SHE4P) protein family of cochaperones, plays an important role in regulating cytoskeletal-associated functions in invertebrates and mammalian cells, including cytokinesis, exocytosis, cell motility, and neuronal development. Here, for the first time, UNC-45A is demonstrated to function as a mitotic spindle-associated protein that destabilizes microtubules (MT) activity. Using in vitro biophysical reconstitution and total internal reflection fluorescence microscopy analysis, we reveal that UNC-45A directly binds to taxol-stabilized MTs in the absence of any additional cellular cofactors or other MT-associated proteins and acts as an ATP-independent MT destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles, and its depletion causes severe defects in chromosome congression and segregation. UNC-45A is overexpressed in human clinical specimens from chemoresistant ovarian cancer and that UNC-45A-overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and restores sensitivity to paclitaxel. IMPLICATIONS: These findings reveal novel and significant roles for UNC-45A in regulation of cytoskeletal dynamics, broadening our understanding of the basic mechanisms regulating MT stability and human cancer susceptibility to paclitaxel, one of the most widely used chemotherapy agents for the treatment of human cancers.

A Novel Preclinical Model of Moderate Primary Blast-Induced Traumatic Brain Injury.

Blast-induced traumatic brain injury (bTBI) is the "signature" injury of the recent Iraq and Afghanistan wars. Here, we present a novel method to induce bTBI using shock wave (SW) lithotripsy. Using a lithotripsy machine, Wistar rats (N = 70; 408.3 ± 93 g) received five SW pulses to the right side of the frontal cortex at 24 kV and a frequency of 60 Hz. Animals were then randomly divided into three study endpoints: 24 h (n = 25), 72 h (n = 19) and 168 h (n = 26). Neurological and behavioral assessments (Garcia's test, beam walking, Rotarod, and elevated plus maze) were performed at the baseline, and further assessments followed at 3, 6, 24, 72, and 168 h post-injury, if applicable. We performed digital subtraction angiography (DSA) to assess presence of cerebral vasospasm due to induced bTBI. Damage to brain tissue was assessed by an overall histological severity (OHS) score based on depth of injury, area of hemorrhage, and extent of axonal injury. Except for beam walking, OHS was significantly correlated with the other three outcome measures with at least one of their assessments during the first 6 h after the experiment. OHS manifested the highest absolute correlation coefficients with anxiety at the baseline and 6 h post-injury (r(baseline) = -0.75, r(6hrs) = 0.85; p<0.05). Median hemispheric differences for contrast peak values (obtained from DSA studies) for 24, 72, and 168 h endpoints were 3.45%, 3.05% and 0.2%, respectively, with statistically significant differences at 1 versus 7 d (p<0.05) and 3 versus 7 d (p<0.01). In this study, we successfully established a preclinical rat model of bTBI with characteristics similar to those observed in clinical cases. This new method may be useful for future investigations aimed at understanding bTBI pathophysiology.

Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.

PURPOSE: Ovarian cancer is the deadliest of the gynecologic malignancies. Carcinogenic progression is accompanied by upregulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUB) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. EXPERIMENTAL DESIGN: The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action were evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intraperitoneal ES-2 xenograft model of human ovarian cancer. RESULTS: Here, we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an unfolded protein response as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival, and was well tolerated by the host. CONCLUSIONS: Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic.

Vaccination for invasive canine meningioma induces in situ production of antibodies capable of antibody-dependent cell-mediated cytotoxicity.

Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ-elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials.