A 14-day pulse of PLX5622 modifies α-synucleinopathy in preformed fibril-infused aged mice of both sexes.

Reactive microglia are observed with aging and in Lewy body disorders, including within the olfactory bulb of men with Parkinson's disease. However, the functional impact of microglia in these disorders is still debated. Resetting these reactive cells by a brief dietary pulse of the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 may hold therapeutic potential against Lewy-related pathologies. To our knowledge, withdrawal of PLX5622 after short-term exposure has not been tested in the preformed α-synuclein fibril (PFF) model, including in aged mice of both sexes. Compared to aged female mice, we report that aged males on the control diet showed higher numbers of phosphorylated α-synuclein+ inclusions in the limbic rhinencephalon after PFFs were injected in the posterior olfactory bulb. However, aged females displayed larger inclusion sizes compared to males. Short-term (14-day) dietary exposure to PLX5622 followed by control chow reduced inclusion numbers and levels of insoluble α-synuclein in aged males-but not females-and unexpectedly raised inclusion sizes in both sexes. Transient delivery of PLX5622 also improved spatial reference memory in PFF-infused aged mice, as evidenced by an increase in novel arm entries in a Y-maze. Superior memory was positively correlated with inclusion sizes but negatively correlated with inclusion numbers. Although we caution that PLX5622 delivery must be tested further in models of α-synucleinopathy, our data suggest that larger-sized-but fewer-α-synucleinopathic structures are associated with better neurological outcomes in PFF-infused aged mice.

α-synucleinopathy exerts sex-dimorphic effects on the multipurpose DNA repair/redox protein APE1 in mice and humans.

Lewy body disorders are characterized by oxidative damage to DNA and inclusions rich in aggregated forms of α-synuclein. Among other roles, apurinic/apyrimidinic endonuclease 1 (APE1) repairs oxidative DNA damage, and APE1 polymorphisms have been linked to cases of Lewy body disorders. However, the link between APE1 and α-synuclein is unexplored. We report that knockdown or inhibition of APE1 amplified inclusion formation in primary hippocampal cultures challenged with preformed α-synuclein fibrils. Fibril infusions into the mouse olfactory bulb/anterior olfactory nucleus (OB/AON) elicited a modest decrease in APE1 expression in the brains of male mice but an increase in females. Similarly, men with Lewy body disorders displayed lower APE1 expression in the OB and amygdala compared to women. Preformed fibril infusions of the mouse OB/AON induced more robust base excision repair of DNA lesions in females than males. No fibril-mediated loss of APE1 expression was observed in male mice when the antioxidant N-acetylcysteine was added to their diet. These findings reveal a potential sex-biased link between α-synucleinopathy and APE1 in mice and humans. Further studies are warranted to determine how this multifunctional protein modifies α-synuclein inclusions and, conversely, how α-synucleinopathy and biological sex interact to modify APE1.

Heat Shock Protein 70 as a Sex-Skewed Regulator of α-Synucleinopathy.

The role of molecular chaperones, such as heat shock protein 70 (Hsp70), is not typically studied as a function of biological sex, but by addressing this gap we might improve our understanding of proteinopathic disorders that predominate in one sex. Therefore, we exposed male or female primary hippocampal cultures to preformed α-synuclein fibrils in a model of early-stage Lewy pathology. We first discovered that two mechanistically distinct inhibitors of Hsp70 function increased phospho-α-synuclein+ inclusions more robustly in male-derived neurons. Because Hsp70 is released into extracellular compartments and may restrict cell-to-cell transmission/amplification of α-synucleinopathy, we then tested the effects of low-endotoxin, exogenous Hsp70 (eHsp70) in primary hippocampal cultures. eHsp70 was taken up by and reduced α-synuclein+ inclusions in cells of both sexes, but pharmacological suppression of Hsp70 function attenuated the inhibitory effect of eHsp70 on perinuclear inclusions only in male neurons. In 20-month-old male mice infused with α-synuclein fibrils in the olfactory bulb, daily intranasal eHsp70 delivery also reduced inclusion numbers and the time to locate buried food. eHsp70 penetrated the limbic system and spinal cord of male mice within 3 h but was cleared within 72 h. Unexpectedly, no evidence of eHsp70 uptake from nose into brain was observed in females. A trend towards higher expression of inducible Hsp70-but not constitutive Hsp70 or Hsp40-was observed in amygdala tissues from male subjects with Lewy body disorders compared to unaffected male controls, supporting the importance of this chaperone in human disease. Women expressed higher amygdalar Hsp70 levels compared to men, regardless of disease status. Together, these data provide a new link between biological sex and a key chaperone that orchestrates proteostasis.

Cardiac Arrest Induced by Asphyxia Versus Ventricular Fibrillation Elicits Comparable Early Changes in Cytokine Levels in the Rat Brain, Heart, and Serum.

Background Current postresuscitative care after cardiac arrest (CA) does not address the cause of CA. We previously reported that asphyxial CA (ACA) and ventricular fibrillation CA (VFCA) elicit unique injury signatures. We hypothesized that the early cytokine profiles of the serum, heart, and brain differ in response to ACA versus VFCA. Methods and Results Adult male rats were subjected to 10 minutes of either ACA or VFCA. Naives and shams (anesthesia and surgery without CA) served as controls (n=12/group). Asphyxiation produced an ≈4-minute period of progressive hypoxemia followed by a no-flow duration of ≈6±1 minute. Ventricular fibrillation immediately induced no flow. Return of spontaneous circulation was achieved earlier after ACA compared with VFCA (42±18 versus 105±22 seconds; P<0.001). Brain cytokines in naives were, in general, low or undetectable. Shams exhibited a modest effect on select cytokines. Both ACA and VFCA resulted in robust cytokine responses in serum, heart, and brain at 3 hours. Significant regional differences pinpointed the striatum as a key location of neuroinflammation. No significant differences in cytokines, neuron-specific enolase, S100b, and troponin T were observed across CA models. Conclusions Both models of CA resulted in marked systemic, heart, and brain cytokine responses, with similar degrees of change across the 2 CA insults. Changes in cytokine levels after CA were most pronounced in the striatum compared with other brain regions. These collective observations suggest that the amplitude of the changes in cytokine levels after ACA versus VFCA may not mediate the differences in secondary injuries between these 2 CA phenotypes.

The center of olfactory bulb-seeded α-synucleinopathy is the limbic system and the ensuing pathology is higher in male than in female mice.

At early disease stages, Lewy body disorders are characterized by limbic vs. brainstem α-synucleinopathy, but most preclinical studies have focused solely on the nigrostriatal pathway. Furthermore, male gender and advanced age are two major risk factors for this family of conditions, but their influence on the topographical extents of α-synucleinopathy and the degree of cell loss are uncertain. To fill these gaps, we infused α-synuclein fibrils in the olfactory bulb/anterior olfactory nucleus complex-one of the earliest and most frequently affected brain regions in Lewy body disorders-in 3-month-old female and male mice and in 11-month-old male mice. After 6 months, we observed that α-synucleinopathy did not expand significantly beyond the limbic connectome in the 9-month-old male and female mice or in the 17-month-old male mice. However, the 9-month-old male mice had developed greater α-synucleinopathy, smell impairment and cell loss than age-matched females. By 10.5 months post-infusion, fibril treatment hastened mortality in the 21.5-month-old males, but the inclusions remained centered in the limbic system in the survivors. Although fibril infusions reduced the number of cells expressing tyrosine hydroxylase in the substantia nigra of young males at 6 months post-infusion, this was not attributable to true cell death. Furthermore, mesencephalic α-synucleinopathy, if present, was centered in mesolimbic circuits (ventral tegmental area/accumbens) rather than within strict boundaries of the nigral pars compacta, which were defined here by tyrosine hydroxylase immunolabel. Nonprimate models cannot be expected to faithfully recapitulate human Lewy body disorders, but our murine model seems reasonably suited to (i) capture some aspects of Stage IIb of Lewy body disorders, which displays a heavier limbic than brainstem component compared to incipient Parkinson's disease; and (ii) leverage sex differences and the acceleration of mortality following induction of olfactory α-synucleinopathy.

Critical appraisal of pathology transmission in the α-synuclein fibril model of Lewy body disorders.

Lewy body disorders are characterized by the emergence of α-synucleinopathy in many parts of the central and peripheral nervous systems, including in the telencephalon. Dense α-synuclein+ pathology appears in regio inferior of the hippocampus in both Parkinson's disease and dementia with Lewy bodies and may disturb cognitive function. The preformed α-synuclein fibril model of Parkinson's disease is growing in use, given its potential for seeding the self-propagating spread of α-synucleinopathy throughout the mammalian brain. Although it is often assumed that the spread occurs through neuroanatomical connections, this is generally not examined vis-à-vis the uptake and transport of tract-tracers infused at precisely the same stereotaxic coordinates. As the neuronal connections of the hippocampus are historically well defined, we examined the first-order spread of α-synucleinopathy three months following fibril infusions centered in the mouse regio inferior (CA2+CA3), and contrasted this to retrograde and anterograde transport of the established tract-tracers FluoroGold and biotinylated dextran amines (BDA). Massive hippocampal α-synucleinopathy was insufficient to elicit memory deficits or loss of cells and synaptic markers in this model of early disease processes. However, dense α-synuclein+ inclusions in the fascia dentata were negatively correlated with memory capacity. A modest compensatory increase in synaptophysin was evident in the stratum radiatum of cornu Ammonis in fibril-infused animals, and synaptophysin expression correlated inversely with memory function in fibril but not PBS-infused mice. No changes in synapsin I/II expression were observed. The spread of α-synucleinopathy was somewhat, but not entirely consistent with FluoroGold and BDA axonal transport, suggesting that variables other than innervation density also contribute to the materialization of α-synucleinopathy. For example, layer II entorhinal neurons of the perforant pathway exhibited somal α-synuclein+ inclusions as well as retrogradely labeled FluoroGold+ somata. However, some afferent brain regions displayed dense retrograde FluoroGold label and no α-synuclein+ inclusions (e.g. medial septum/diagonal band), supporting the selective vulnerability hypothesis. The pattern of inclusions on the contralateral side was consistent with specific spread through commissural connections (e.g. stratum pyramidale of CA3), but again, not all commissural projections exhibited α-synucleinopathy (e.g. hilar mossy cells). The topographical extent of inclusions is displayed here in high-resolution images that afford viewers a rich opportunity to dissect the potential spread of pathology through neural circuitry. Finally, the results of this expository study were leveraged to highlight the challenges and limitations of working with preformed α-synuclein fibrils.

Evidence for cross-hemispheric preconditioning in experimental Parkinson's disease.

Dopamine loss and motor deficits in Parkinson's disease typically commence unilaterally and remain asymmetric for many years, raising the possibility that endogenous defenses slow the cross-hemispheric transmission of pathology. It is well-established that the biological response to subtoxic stress prepares cells to survive subsequent toxic challenges, a phenomenon known as preconditioning, tolerance, or stress adaptation. Here we demonstrate that unilateral striatal infusions of the oxidative toxicant 6-hydroxydopamine (6-OHDA) precondition the contralateral nigrostriatal pathway against the toxicity of a second 6-OHDA infusion in the opposite hemisphere. 6-OHDA-induced loss of dopaminergic terminals in the contralateral striatum was ablated by cross-hemispheric preconditioning, as shown by two independent markers of the dopaminergic phenotype, each measured by two blinded observers. Similarly, loss of dopaminergic somata in the contralateral substantia nigra was also abolished, according to two blinded measurements. Motor asymmetries in floor landings, forelimb contacts with a wall, and spontaneous turning behavior were consistent with these histological observations. Unilateral 6-OHDA infusions increased phosphorylation of the kinase ERK2 and expression of the antioxidant enzyme CuZn superoxide dismutase in both striata, consistent with our previous mechanistic work showing that these two proteins mediate preconditioning in dopaminergic cells. These findings support the existence of cross-hemispheric preconditioning in Parkinson's disease and suggest that dopaminergic neurons mount impressive natural defenses, despite their reputation as being vulnerable to oxidative injury. If these results generalize to humans, Parkinson's pathology may progress slowly and asymmetrically because exposure to a disease-precipitating insult induces bilateral upregulation of endogenous defenses and elicits cross-hemispheric preconditioning.

Time in therapeutic range as a marker for thrombotic and bleeding outcomes in Fontan patients.

Fontan patients managed with warfarin are at risk not only for thrombotic events, but also for bleeding episodes as a consequence of anticoagulation treatment. The aim of this study was to determine whether time spent in patient specified therapeutic range (TTR), when managed in a cardiology-based pharmacist managed anticoagulation clinic (PMAC), is a useful target metric for monitoring, as well as improving outcomes. A single center retrospective review was conducted evaluating TTR of all Fontan patients (n = 45) on warfarin managed in our outpatient cardiology pharmacist managed anticoagulation clinic (PMAC) during a 19 month time frame. The primary outcome was time spent within, above, and below therapeutic range. Secondary outcomes were thrombotic event (TE) incidence pre- and post PMAC enrollment and bleeding event incidence during PMAC management. Of the Fontan patients included, 55.6% were male and the median age at latest anticoagulation clinic follow-up was 19 years (IQR 13, 29). A composite 52.9 patient years of warfarin therapy was evaluated during the study time frame. The mean TTR for patients was 84.1 ± 5.2%. The most frequent reasons for non-therapeutic INRs were diet changes (42.8%), medication non-compliance (13.7%), and drug interactions (8.8%). Only one TE occurred during the study time frame. The incidence of TE in this population was decreased after PMAC enrollment (1 per 52.9 patient year versus 1 event per 17.4 patient year; p < 0.0002). Two major bleeds that required emergency department visit occurred during this time, none were cerebral or gastrointestinal. In Fontan patients anticoagulated with warfarin, a greater than 80% TTR can be achieved in a PMAC. Such high time in therapeutic range was associated with excellent outcomes, despite the obvious complexity of this population.

Transmission of α-synucleinopathy from olfactory structures deep into the temporal lobe.

BACKGROUND: α-synucleinopathy emerges quite early in olfactory structures such as the olfactory bulb and anterior olfactory nucleus (OB/AON) in Parkinson's disease. This may contribute to smell impairments years before the commencement of motor symptoms. We tested whether α-synucleinopathy can spread from the OB/AON to regions of the limbic telencephalon that harbor connections with olfactory structures. FINDINGS: α-synuclein fibrils were infused into the OB/AON. Inclusions containing pathologically phosphorylated α-synuclein (pSer129) were observed three months later in the piriform and entorhinal cortices, amygdala, and hippocampal formation. The retrograde tract-tracer FluoroGold confirmed the existence of first-order afferents at these sites. Some sites harbored FluoroGold(+) neurons but no inclusions, suggestive of selective vulnerabilities. Multiple areas close to the injection site but not connected with the OB/AON remained free of inclusions, suggesting a lack of widespread uptake of fibrils from interstitial diffusion. Two independent pSer129 antibodies revealed the same labeling patterns and preadsorption control experiments confirmed a loss of pSer129 staining. Dense total α-synuclein (but not pSer129) staining was apparent in the OB/AON 1.5 h following fibril infusions, suggesting that pSer129(+) staining did not reflect exogenously infused material. Waterbath sonication of fibrils for 1 h improved α-synucleinopathy transmission relative to 1 min-long probe sonication. Electron microscopy revealed that longer sonication durations reduced fibril size. The Thioflavin stain labeled cells at the infusion site and some, but not all inclusions contained ubiquitin. Three-dimensional confocal analyses revealed that many inclusions ensconced NeuN(+) neuronal nuclei. Young and aged mice exhibited similar topographical spread of α-synucleinopathy. CONCLUSIONS: 1) α-synucleinopathy in this model is transmitted through some, but not all neuroanatomical connections, 2) pathology is largely confined to first-order afferent sites at three months and this is most parsimoniously explained by retrograde transport, and 3) transmission in aged animals is largely similar to that in young control animals at three months post-infusion.