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.

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.