Autophagy and protein aggregation as a mechanism of dopaminergic degeneration in a primary human dopaminergic neuronal model.

The pathophysiology underlying the loss of dopaminergic neurons in Parkinson's disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0-10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.

Tauroursodeoxycholic acid (TUDCA) is neuroprotective in a chronic mouse model of Parkinson's disease.

OBJECTIVE: Parkinson's disease (PD) is a progressive motor disease of unknown etiology. Although neuroprotective ability of endogenous bile acid, tauroursodeoxycholic acid (TUDCA), shown in various diseases, including an acute model of PD,the potential therapeutic role of TUDCA in progressive models of PD that exhibit all aspects of PD has not been elucidated. In the present study, mice were assigned to one of four treatment groups: (1) Probenecid (PROB); (2) TUDCA, (3) MPTP + PROB (MPTPp); and (3) TUDCA + MPTPp. Methods: Markers for dopaminergic function, neuroinflammation, oxidative stress and autophagy were assessed using high performance liquid chromatography (HPLC), immunohistochemistry (IHC) and western blot (WB) methods. Locomotion was measured before and after treatments. Results: MPTPp decreased the expression of dopamine transporters (DAT) and tyrosine hydroxylase (TH), indicating dopaminergic damage, and induced microglial and astroglial activation as demonstrated by IHC analysis. MPTPp also decreased DA and its metabolites as demonstrated by HPLC analysis. Further, MPTPp-induced protein oxidation; increased LAMP-1 expression indicated autophagy and the promotion of alpha-synuclein (α-SYN) aggregation. Discussion: Pretreatment with TUDCA protected against dopaminergic neuronal damage, prevented the microglial and astroglial activation, as well as the DA and DOPAC reductions caused by MPTPp. TUDCA by itself did not produce any significant change, with data similar to the negative control group. Pretreatment with TUDCA prevented protein oxidation and autophagy, in addition to inhibiting α-SYN aggregation. Although TUDCA pretreatment did not significantly affect locomotion, only acute treatment effects were measured, indicating more extensive assessments may be necessary to reveal potential therapeutic effects on behavior. Together, these results suggest that autophagy may be involved in the progression of PD and that TUDCA may attenuate these effects. The efficacy of TUDCA as a novel therapy in patients with PD clearly warrants further study.

Genotoxicity evaluation using primary hepatocytes isolated from rhesus macaque (Macaca mulatta).

Non-human primates (NHPs) have played a vital role in fundamental, pre-clinical, and translational studies because of their high physiological and genetic similarity to humans. Here, we report a method to isolate primary hepatocytes from the livers of rhesus macaques (Macaca mulatta) after in situ whole liver perfusion. Isolated primary macaque hepatocytes (PMHs) were treated with various compounds known to have different pathways of genotoxicity/carcinogenicity and the resulting DNA damage was evaluated using the high-throughput CometChip assay. The comet data were quantified using benchmark dose (BMD) modeling and the BMD50 values for treatments of PMHs were compared with those generated from primary human hepatocytes (PHHs) in our previous study (Seo et al. Arch Toxicol 2020, 2207-2224). The results showed that despite varying CYP450 enzyme activities, PMHs had the same sensitivity and specificity as PHHs in detecting four indirect-acting (i.e., requiring metabolic activation) and seven direct-acting genotoxicants/carcinogens, as well as five non-carcinogens that are negative or equivocal for genotoxicity in vivo. The BMD50 estimates and their confidence intervals revealed species differences for DNA damage potency, especially for direct-acting compounds. The present study provides a practical method for maximizing the use of animal tissues by isolating primary hepatocytes from NHPs. Our data support the use of PMHs as a reliable surrogate of PHHs for evaluating the genotoxic hazards of chemical substances for humans.

Can SARS-CoV-2 infect the central nervous system via the olfactory bulb or the blood-brain barrier?

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China in December 2019. On February 11, the World Health Organization (WHO) announced the name for the new illness caused by SARS-CoV-2: COVID-19. By March 11, the outbreak of COVID-19 was declared a pandemic by the WHO. This virus has extensively altered daily life for many across the globe, while claiming hundreds of thousands of lives. While fundamentally a respiratory illness, many infected individuals experience symptoms that involve the central nervous system (CNS). It is likely that many of these symptoms are the result of the virus residing outside of the CNS. However, the current evidence does indicate that the SARS-CoV-2 virus can use olfactory neurons (or other nerve tracts) to travel from the periphery into the CNS, and that the virus may also enter the brain through the blood-brain barrier (BBB). We discuss how the virus may use established infection mechanisms (ACE2, NRP1, TMPRSS2, furin and Cathepsin L), as well mechanisms still under consideration (BASIGIN) to infect and spread throughout the CNS. Confirming the impact of the virus on the CNS will be crucial in dealing with the long-term consequences of the epidemic.

Regions of the basal ganglia and primary olfactory system are most sensitive to neurodegeneration after extended sevoflurane anesthesia in the perinatal rat.

Extended general anesthesia early in life is neurotoxic in multiple species. However, little is known about the temporal progression of neurodegeneration after general anesthesia. It is also unknown if a reduction in natural cell death, or an increase in cell creation, occurs as a form of compensation after perinatal anesthesia exposure. The goal of this study was to evaluate markers of neurodegeneration and cellular division at 2, 24, or 72 h after sevoflurane (Sevo) exposure (6 h) in fully oxygenated postnatal day (PND) 7 rats. Neurodegeneration was observed in areas throughout the forebrain, while the largest changes (fold increase above vehicle) were observed in areas associated with either the primary olfactory learning pathways or the basal ganglia. These regions included the indusium griseum (IG, 25-fold), the posterior dorso medial hippocampal CA1 (17-fold), bed nucleus of the stria terminalis (Bed Nuclei STM, 5-fold), the shell of the nucleus accumbens (Acb, 5-fold), caudate/putamen (CPu, 5-fold), globus pallidus (GP, 9-fold) and associated thalamic (11-fold) and cortical regions (5-fold). Sevo neurodegeneration was minimal or undetectable in the ventral tegmentum, substantia nigra, and most of the hypothalamus and frontal cortex. In most brain regions where neurodegeneration was increased 2 h post Sevo exposure, the levels returned to <4-fold above control levels by 24 h. However, in the IG, CA1, GP, anterior thalamus, medial preoptic nucleus of the hypothalamus (MPO), anterior hypothalamic area (AHP), and the amygdaloid nuclei, neurodegeneration at 24 h was double or more than that at 2 h post exposure. Anesthesia exposure causes either a prolonged period of neurodegeneration in certain brain regions, or a distinct secondary degenerative event occurs after the initial insult. Moreover, regions most sensitive to Sevo neurodegeneration did not necessarily coincide with areas of new cell birth, and new cell birth was not consistently affected by Sevo. The profile of anesthesia related neurotoxicity changes with time, and multiple mechanisms of toxicity may exist in a time-dependent fashion.

Microglial activation and responses to vasculature that result from an acute LPS exposure.

Bacterial cell wall endotoxins, i.e. lipopolysaccharides (LPS), are some of the original compounds shown to evoke the classic signs of systemic inflammation/innate immune response and neuroinflammation. The term neuroinflammation often is used to infer the elaboration of proinflammatory mediators by microglia elicited by neuronal targeted activity. However, it also is possible that the microglia are responding to vasculature through several signaling mechanisms. Microglial activation relative to the vasculature in the hippocampus and parietal cortex was determined after an acute exposure of a single subcutaneous injection of 2 mg/kg LPS. Antibodies to allograft inflammatory factor (Aif1, a.k.a. Iba1) were used to track and quantify morphological changes in microglia. Immunostaining of platelet/endothelial cell adhesion molecule 1 (Pecam1, a.k.a. Cd31) was used to visualize vasculature in the forebrain and glial acidic fibrillary protein (GFAP) to visualize astrocytes. Neuroinflammation and other aspects of neurotoxicity were evaluated histologically at 3 h, 6 h, 12 h, 24 h, 3 d and 14 d following LPS exposure. LPS did not cause neurodegeneration as determined by Fluoro Jade C labeling. Also, there were no signs of mouse IgG leakage from brain vasculature due to LPS. Some changes in microglia size occurred at 6 h, but by 12 h microglial activation had begun with the combined soma and proximal processes size increasing significantly (1.5-fold). At 24 h, almost all the microglia soma and proximal processes in the hippocampus, parietal cortex, and thalamus were closely associated with the vasculature and had increased almost 2.0-fold in size. In many areas where microglia were juxtaposed to vasculature, astrocytic endfeet appeared to be displaced. The microglial activation had subsided slightly by 3 d with microglial size 1.6-fold that of control. We hypothesize that acute LPS activation can result in vascular mediated microglial responses through several mechanisms: 1) binding to Cd14 and Tlr4 receptors on microglia processes residing on vasculature; 2) damaging vasculature and causing the release of cytokines; and 3) possibly astrocytic endfeet damage resulting in cytokine release. These acute responses may serve as an adaptive mechanism to exposure to circulating LPS where the microglia surround the vasculature. This could further prevent the pathogen(s) circulating in blood from entering the brain. However, diverting microglial interactions away from synaptic remodeling and other types of microglial interactions with neurons may have adverse effects on neuronal function.

Stretch-Induced Deformation as a Model to Study Dopaminergic Dysfunction in Traumatic Brain Injury.

Traumatic brain injury (TBI) is defined as damage to the brain that consequently disrupts normal function. Neuronal death, a hallmark of TBI, has been related to the development of neurodegenerative disorders like Parkinson's disease (PD), where loss of dopaminergic neurons and dopaminergic dysfunction are observed. To date, no in vitro model exists in which the dopaminergic damage observed in TBI is replicated. In this study, we evaluated the effects of in vitro simulated TBI on human dopaminergic neurons. To simulate TBI, neurons were subjected to 0%, 5%, 10%, 15%, 25% and 50% deformation. 24 h after injury, cell viability and apoptosis were determined by lactate dehydrogenase (LDH) release and DNA fragmentation, as well as ethidium homodimer and caspase 3/7 staining. Dopamine (DA) levels were determined by ELISA. Levels of tyrosine hydroxylase (TH) and DA transporter (DAT) were determined by western blot. Only 50% stretch increased LDH release and ethidium homodimer staining, suggesting the induction of necrosis. On the contrary, 25% and 50% stretch increased DNA fragmentation while 15%, 25% and 50% increased caspase 3/7 staining, suggesting that moderate and severe TBI promote apoptosis. Levels of intracellular DA decreased in a stretch-dependent manner with 15%, 25% and 50% stretch, which were related with a decrease in TH expression. Extracellular DA levels increased only at 50%. Levels of DAT remained unchanged regardless of treatment. These data support the use of stretch as a model to simulate TBI in vitro in human dopaminergic neurons, replicating the acute effects of TBI in the dopaminergic system.

Amyloid Beta 25-35 induces blood-brain barrier disruption in vitro.

The amyloid ß-peptide (Aß) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aß fraction 25-35 (Aß25-35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aß with RAGE compromises the BBB function in Alzheimer' disease (AD). Here, we evaluated the effects of Aß25-35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20 µM active Aß25-35 or the inactive Aß35-25 (control), for 24 h. Exposure to Aß25-35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aß25-35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aß25-35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aß25-35-induces toxicity by compromising the functionality and integrity of the BBB in vitro. Graphical abstract Aß25-35 induces BBB dysfunction in vitro, wich is likely mediated by OS and ultimately leads to disruption of BBB integrity and cell death.

Neuroprotective effects of acetyl-l-carnitine (ALC) in a chronic MPTP-induced Parkinson's disease mouse model: Endothelial and microglial effects.

Parkinson's disease (PD) is a progressive motor disease with clinical features emerging due to degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which project to the caudate putamen (CPu) where they release dopamine (DA). The current study investigated whether acetyl-l-carnitine (ALC) could ameliorate the pathology seen in an in vivoin vivo chronic 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of PD. Four treatment groups were included: 1) CONTROL receiving probenecid (PROB; 250 mg/kg) only, 2) MPTP (25 mg/kg) + PROB, 3) MPTP + ALC (100 mg/kg), and 4) ALC alone. MPTP-induced losses in tyrosine hydroxylase and DA transporter immunoreactivity in the SNc and CPu were significantly reduced by ALC. HPLC data further suggests that decreases in CPu DA levels produced by MPTP were also attenuated by ALC. Additionally, microglial activation and astrocytic reactivity induced by MPTP were greatly reduced by ALC, indicating protection against neuroinflammation. Glucose transporter-1 and the tight junction proteins occludin and zonula occludins-1 were also protected from MPTP-induced down-regulation by ALC. Together, data suggest that in this model, ALC protects against MPTP-induced damage to endothelial cells and loss of DA neurons in the SNc and CPu, suggesting that ALC therapy may have the potential to slow or ameliorate the progression of PD pathology in a clinical setting.

'The environment matters' and 'designing toward the whole'.

A nurse/health care executive/architecture specialist and an artist/designer offer insights to creating environments that effectively support the delivery of care, the wellbeing of patients and their families, and the well-being of the health care workforce. The care environment is more than bricks, mortar, doors, windows, walls, and flooring. It is a sanctuary where healing can take place, where the spirit can be released, and where the provision of care, often fairly intimate, can happen through the connection between patient and care providers. Evidence-based design has brought forth the kind of inquiry and solutions that begin to address what an environment for healing requires. The interaction of built environment, staff, patients, families, and doctors is an overlapping ebb and flow that relies at its heart on the ability to hold all the parts as having equal priority.