The Synucleins and the Astrocyte.

Synucleins consist of three proteins exclusively expressed in vertebrates. α-Synuclein (αS) has been identified as the main proteinaceous aggregate in Lewy bodies, a pathological hallmark of many neurodegenerative diseases. Less is understood about ß-synuclein (ßS) and γ-synuclein (γS), although it is known ßS can interact with αS in vivo to inhibit aggregation. Likewise, both γS and ßS can inhibit αS's propensity to aggregate in vitro. In the central nervous system, ßS and αS, and to a lesser extent γS, are highly expressed in the neural presynaptic terminal, although they are not strictly located there, and emerging data have shown a more complex expression profile. Synapse loss and astrocyte atrophy are early aspects of degenerative diseases of the brain and correlate with disease progression. Synucleins appear to be involved in synaptic transmission, and astrocytes coordinate and organize synaptic function, with excess αS degraded by astrocytes and microglia adjacent to the synapse. ßS and γS have also been observed in the astrocyte and may provide beneficial roles. The astrocytic responsibility for degradation of αS as well as emerging evidence on possible astrocytic functions of ßS and γS, warrant closer inspection on astrocyte-synuclein interactions at the synapse.

Astrocytes Imagined.

The cellular, molecular and physiological basis of cognition has proved elusive until emerging studies on astrocytes. The appearance of a deliberate aggregating element in cellular neurophysiology was difficult to satisfy computationally with excitatory and inhibitory neuron physiology alone. Similarly, the complex behavioral outputs of cognition are challenging to test experimentally. Astrocytic reception and control of synaptic communication has provided the possibility for study of the missing element. The advancement of genetic and neurophysiological techniques have now demonstrated astrocytes respond to neural input and subsequently provide the ability for neural synchronization and assembly at multiple and single synaptic levels. Considering the most recent evidence, it is becoming clear that astrocytes contribute to cognition. Is it possible then that our cognitive experience is essentially the domain of astrocyte physiology, ruminating on neural input, and controlling neural output? Although the molecular and cellular complexities of cognition in the human nervous system cannot be overstated, in order to gain a better understanding of the current evidence, an astrocyte centric basis of cognition will be considered from a philosophical, biological and computational perspective.

The Astrogenic Balance in the Aging Brain.

An inverse correlation between the incidence of cancer and neurodegenerative disease has been observed, with the prevalence of cancer peaking around 60 years of age, then slowly tapering off as neurodegenerative diseases increase in the elderly. Although the diseases rarely occur concurrently, the same genes are differentially expressed between the diseases, with four transcription factors found to be in common for their expression. In the brain, mature astrocytes are the origin of astrocytoma, which make up 58.2% of malignant brain tumors in patients 65 or older, while GFAP+ astrocyte-like neural stem cells from the subventricular zone give rise to glioblastoma and anaplastic astrocytoma, which make up 41.6%. Likewise, in neurodegenerative disease, a decrease in astrocyte density is observed in early disease states, and senescent astrocytes increase. Because astrocytes coordinate synaptic function, astrocyte dysfunction likely contributes to or causes initial synapse loss and cognitive decline seen in neurodegenerative disease. In non-disease states, astrocytes retain their ability to successfully re-enter the cell cycle through adult astrogenesis to maintain the neuroenvironment, and controlled astrocytic proliferation could be an important contributor to neurological function. Disruption to this astrogenic balance could account for the inverse correlation of cell cycle dysregulation resulting in malignant astrocytes and tumorigenesis, and astrocytic senescence and cell death without self-renewal in aging resulting in neurodegenerative disease. The current understanding of the astrocytic roles of the transcription factors that could be the cause of this imbalance will be discussed, as well as possible therapeutic approaches to modulate their expression in the astrocyte.

DRD4 Allele Frequency as a Lab Exercise in Neuroscience and Genetics Courses.

DNA segments with variable number tandem repeats (VNTR) serve as a model for students to learn DNA extraction and polymerase chain reaction (PCR) techniques in biology laboratory courses from the high school to the graduate level. Because of a growing interest in the neurosciences among undergraduates, we have developed a PCR exercise with a focus on the nervous system and behavior, with the aim of inspiring students from all aspects of the neurosciences to appreciate the central dogma and neurogenetics. DRD4 was a good candidate to provide a lab exercise that would be more engaging than VNTR analysis of a non-coding segment. DRD4 encodes for the dopamine D4 receptor and has been controversially associated with 'novelty seeking' or 'wanderlust' behavior. DRD4 has three common variants of the 48 bp sequence on exon 3, easily differentiated through gel electrophoresis. The 2 repeat (2R), 4 repeat (4R) and 7 repeat (7R) of the 48 bp sequence are the most common alleles. The 7R sequences result in the expressed dopamine D4 receptor having less affinity for dopamine binding, which was proposed to be the reason individuals engage in 'novelty seeking' behavior, to increase dopamine release to facilitate more binding to the receptor. Here we demonstrate an enjoyable and simple two lab sequence to analyze DRD4 genotypes that is appropriate for neuroscience and genetics courses.

Chimera RNA interference knockdown of γ-synuclein in human cortical astrocytes results in mitotic catastrophe.

Elevated levels of γ-synuclein (γ-syn) expression have been noted in the progression of glioblastomas, and also in the cerebrospinal fluid of patients diagnosed with neurodegenerative diseases. γ-Syn can be either internalized from the extracellular milieu or expressed endogenously by human cortical astrocytes. Internalized γ-syn results in increased cellular proliferation, brain derived neurotrophic factor release and astroprotection. However, the function of endogenous γ-syn in primary astrocytes, and the relationship to these two opposing disease states are unknown. γ-Syn is expressed by astrocytes in the human cortex, and to gain a better understanding of the role of endogenous γ-syn, primary human cortical astrocytes were treated with chimera RNA interference (RNAi) targeting γ-syn after release from cell synchronization. Quantitative polymerase chain reaction analysis demonstrated an increase in endogenous γ-syn expression 48 hours after release from cell synchronization, while RNAi reduced γ-syn expression to control levels. Immunocytochemistry of Ki67 and 5-bromodeoxyuridine showed chimera RNAi γ-syn knockdown reduced cellular proliferation at 24 and 48 hours after release from cell synchronization. To further investigate the consequence of γ-syn knockdown on the astrocytic cell cycle, phosphorylated histone H3 pSer10 (pHH3) and phosphorylated cyclin dependent kinase-2 pTyr15 (pCDK2) levels were observed via western blot analysis. The results revealed an elevated expression of pHH3, but not pCDK2, indicating γ-syn knockdown leads to disruption of the cell cycle and chromosomal compaction after 48 hours. Subsequently, flow cytometry with propidium iodide determined that increases in apoptosis coincided with γ-syn knockdown. Therefore, γ-syn exerts its effect to allow normal astrocytic progression through the cell cycle, as evidenced by decreased proliferation marker expression, increased pHH3, and mitotic catastrophe after knockdown. In this study, we demonstrated that the knockdown of γ-syn within primary human cortical astrocytes using chimera RNAi leads to cell cycle disruption and apoptosis, indicating an essential role for γ-syn in regulating normal cell division in astrocytes. Therefore, disruption to γ-syn function would influence astrocytic proliferation, and could be an important contributor to neurological diseases.

γ-Synuclein Induces Human Cortical Astrocyte Proliferation and Subsequent BDNF Expression and Release.

γ-Synuclein (γ-syn) is expressed by astrocytes in the human nervous system, and increased extracellularly in the brain and cerebrospinal fluid of individuals diagnosed with Alzheimer's disease. Upregulation of γ-syn also coincides with proliferation of glioblastomas and other cancers. In order to better understand regulation and function of extracellular γ-syn, primary human cortical astrocytes were treated with γ-syn conditioned media at various physiological concentrations (50, 100, 150 nM) after cell synchronization. Additionally, extracellular brain-derived neurotrophic factor (BDNF), a neuroprotective growth factor released by astrocytes that has been shown to be decreased extracellularly in neurodegenerative disease, was observed in response to γ-syn treatment. Analysis of 5-bromodeoxyuridine (BrdU) and propidium iodide through flow cytometry 24 h after release from synchronization revealed an increase in G2/M phase of the cell cycle with 100 nM γ-syn during initial cell division, an effect that was reversed at 48 h. However, increased extracellular BDNF was observed at 48 h with 100 nM and 150 nM γ-syn treatment with no difference between controls at 24 h. Further analysis of cell cycle markers with immunocytochemistry of BrdU and Ki67 after treatment with 100 nM γ-syn confirmed increased initial cell proliferation and decreased non-proliferating cells. Western blot analysis demonstrated increased γ-syn levels after 100 nM treatment at 24 and 48 h, and increased pro-BDNF, mature BDNF and cell viability at 48 h. The results demonstrate that γ-syn internalization by human cortical astrocytes causes upregulation of the cell cycle, followed by subsequent BDNF expression and release.

Adult Astrogenesis and the Etiology of Cortical Neurodegeneration.

As more evidence points to a clear role for astrocytes in synaptic processing, synaptogenesis and cognition, continuing research on astrocytic function could lead to strategies for neurodegenerative disease prevention. Reactive astrogliosis results in astrocyte proliferation early in injury and disease states and is considered neuroprotective, indicating a role for astrocytes in disease etiology. This review describes the different types of human cortical astrocytes and the current evidence regarding adult cortical astrogenesis in injury and degenerative disease. A role for disrupted astrogenesis as a cause of cortical degeneration, with a focus on the tauopathies and synucleinopathies, will also be considered.

Neurogranin binds α-synuclein in the human superior temporal cortex and interaction is decreased in Parkinson's disease.

Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson's disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson's disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson's disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson's disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory.

TOM40 mediates mitochondrial dysfunction induced by α-synuclein accumulation in Parkinson's disease.

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson's disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery--TOM40--might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies.

Lovastatin ameliorates alpha-synuclein accumulation and oxidation in transgenic mouse models of alpha-synucleinopathies.

Alpha-synuclein (alpha-syn) aggregation is a neuropathological hallmark of many diseases including Dementia with Lewy Bodies (DLB) and Parkinson's Disease (PD), collectively termed the alpha-synucleinopathies. The mechanisms underlying alpha-syn aggregation remain elusive though emerging science has hypothesized that the interaction between cholesterol and alpha-syn may play a role. Cholesterol has been linked to alpha-synucleinopathies by recent work suggesting cholesterol metabolites appear to accelerate alpha-syn fibrillization. Consistent with these findings, cholesterol-lowering agents have been demonstrated to reduce alpha-syn accumulation and the associated neuronal pathology in vitro. In this context, this study sought to investigate the in vivo effects of the cholesterol synthesis inhibitor lovastatin on alpha-syn aggregation in two different transgenic (Tg) mouse models that neuronally overexpress human alpha-syn. Lovastatin-treated mice displayed significantly reduced plasma cholesterol levels and levels of oxidized cholesterol metabolites in the brain in comparison to saline-treated controls. Immunohistochemical analysis demonstrated a significant reduction of neuronal alpha-syn aggregates and alpha-syn immunoreactive neuropil in the temporal cortex of lovastatin-treated Tg mice in comparison to saline-treated alpha-syn Tg controls. Consistently, immunoblot analysis of mouse brain homogenates showed a reduction in levels of total and oxidized alpha-syn in lovastatin-treated alpha-syn Tg mice in comparison to saline-treated alpha-syn Tg controls. The reduced alpha-syn accumulation in lovastatin-treated mice was associated with abrogation of neuronal pathology. The results from this study demonstrate that lovastatin administration can reduce alpha-syn aggregation and associated neuropathology and support the possibility that treatment with cholesterol-lowering agents may be beneficial for patients with PD and/or DLB.

GFAP reactivity, apolipoprotein E redistribution and cholesterol reduction in human astrocytes treated with alpha-synuclein.

Alpha-synuclein (alpha-syn) is an abundant neuronal protein expressed at the synapse. In neurodegenerative disease alpha-syn accumulates in the extracellular space. Astrocytes present at neural synapses are thought to contribute to synaptogenesis through cholesterol release and normally exhibit increased glial fibrillary acid protein (GFAP) reactivity and apolipoprotein E (apoE) expression in neurodegenerative disease states. We proposed that extracellular alpha-syn treatment of human astrocytes would impact cholesterol levels and expression of GFAP and apolipoprotein E (apoE). Human astrocytes were treated with alpha-syn at different concentrations and time points to determine the effective membrane permeability of the peptide. After alpha-syn treatment, we analyzed apoE and cholesterol levels in the astrocyte membrane. Lastly, we performed immunocytochemistry for GFAP in control and alpha-syn treated cells. Our results indicate membrane apoE was reduced and redistributed from a nuclear and membranous dominated expression to the cytosol. Cholesterol levels were also reduced in the astrocyte cell membrane. GFAP expression was sharply increased in alpha-syn treated cells indicating that alpha-syn may contribute to reactive gliosis. Our results support the conclusion that astrocytes play a role in pathological mechanisms in synucleinopathies.

Behavioral recovery from traumatic brain injury after membrane reconstruction using polyethylene glycol.

Polyethylene glycol (PEG; 2000 MW, 30% by volume) has been shown to mechanically repair damaged cellular membranes and reduce secondary axotomy after traumatic brain and spinal cord injury (TBI and SCI respectively). This repair is achieved following spontaneous reassembly of cell membranes made possible by the action of targeted hydrophilic polymers which first seal the compromised portion of the plasmalemma, and secondarily, allow the lipidic core of the compromised membranes to resolve into each other. Here we compared PEG-treated to untreated rats using a computer-managed open-field behavioral test subsequent to a standardized brain injury. Animals were evaluated after a 2-, 4-, and 6-hour delay in treatment after TBI. Treated animals receive a single subcutaneous injection of PEG. When treated within 2 hours of the injury, injured PEG-treated rats showed statistically significant improvement in their exploratory behavior recorded in the activity box when compared to untreated but brain-injured controls. A delay of 4 hours reduced this level of achievement, but a statistically significant improvement due to PEG injection was still clearly evident in most outcome measures compared at the various evaluation times. A further delay of 2 more hours, however, eradicated the beneficial effects of PEG injection as revealed using this behavioral assessment. Thus, there appears to be a critical window of time in which PEG administration after TBI can provide neuroprotection resulting in an enhanced functional recovery. As is often seen in clinically applied acute treatments for trauma, the earlier the intervention can be applied, the better the outcome.

Cellular genesis in the postnatal piglet.

Because of the anatomical and developmental similarity of the piglet brain to the human brain we were interested in characterizing the areas of cellular genesis which occur postnatally to validate the model for subsequent neurobiological research. In this study, four piglets were injected with 5-bromodeoxyuridine (BrdU) at 6, 7 and 8 days of age. The animals were sacrificed at 13 days of age and the brains were analyzed to characterize areas of cellular genesis. BrdU was seen throughout the brain and found to be most abundant in the subventricular zone (SVZ); doublecortin (DCX) expressing cells were found throughout the white matter-with an extensive DCX network in the SVZ. Here we describe for the first time the use of immunohistochemistry for BrdU and DCX to study cellular genesis in the piglet brain.

Statins reduce neuronal alpha-synuclein aggregation in in vitro models of Parkinson's disease.

Aggregation of alpha-synuclein (alpha-syn) is believed to play a critical role in the pathogenesis of disorders such as dementia with Lewy bodies and Parkinson's disease. The function of alpha-syn remains unclear, although several lines of evidence suggest that alpha-syn is involved in synaptic vesicle trafficking probably via lipid binding. Moreover, interactions with cholesterol and lipids have been shown to be involved in alpha-syn aggregation. In this context, the main objective of this study was to determine if statins--cholesterol synthesis inhibitors--might interfere with alpha-syn accumulation in cellular models. For this purpose, we studied the effects of lovastatin, simvastatin, and pravastatin on the accumulation of alpha-syn in a stably transfected neuronal cell line and in primary human neurons. Statins reduced the levels of alpha-syn accumulation in the detergent insoluble fraction of the transfected cells. This was accompanied by a redistribution of alpha-syn in caveolar fractions, a reduction in oxidized alpha-syn, and enhanced neurite outgrowth. In contrast, supplementation of the media with cholesterol increased alpha-syn aggregation in detergent insoluble fractions of transfected cells and was accompanied by reduced neurite outgrowth. Taken together, these results suggest that regulation of cholesterol levels with cholesterol inhibitors might be a novel approach for the treatment of Parkinson's disease.

A novel open field activity detector to determine spatial and temporal movement of laboratory animals after injury and disease.

The wide range of tests for laboratory animal behavior after neurological injury or disease each have their benefits and detriments. The varied behavior an animal exhibits makes it difficult to decide which test to use. However, a fundamental instinct for the laboratory animal is to explore when placed in a new environment. A way to test exploratory behavior is in the open field. Here, we introduce a simple activity box without the use of video equipment to determine the exploratory movement of a rat after traumatic brain injury. The activity box is an open field and the rat explores its surroundings when placed inside. Four infrared beams were placed in both the X and Y-axis inside the box. Using a novel system to determine which beam the rat breaks, we describe where the rat is in space and time while in the activity box. Other models can show the number of beams broken, but here we elucidate the methods to additionally determine the amount of area explored, the total distance traveled by the rat and percent time exploring.

Polyethylene glycol treatment after traumatic brain injury reduces beta-amyloid precursor protein accumulation in degenerating axons.

Polyethylene glycol (PEG; 2,000 MW; 30% v/v) is a nontoxic molecule that can be injected intravenously and possesses well-documented neuroprotective properties in the spinal cord of the guinea pig. Recent studies have shown that intravenous PEG can also enter the rat brain parenchyma after injury and repair cellular membrane damage in the region of the corpus callosum. Disrupted anterograde axonal transport and resulting beta-amyloid precursor protein (APP) accumulation are byproducts of traumatic axonal injury (TAI) in the brain. APP accumulation indicates axonal degeneration as a result of axotomy, a detriment that can lead to cell death. In this study, we show that PEG treatment can eliminate APP accumulation in specific brain areas of rats receiving TAI. Six areas of the brain were analyzed: the medial cortex, hippocampus, lateral cortex, thalamus, medial lemniscus, and medial longitudinal fasciculus. Increased APP expression after injury was abolished in the thalamus and reduced in the medial longitudinal fasciculus by PEG treatment. In all remaining areas except for the lateral cortex, APP expression was not increased between injured and uninjured brains, indicating that damage was undetected in those brain areas in this study.

Intravenous polyethylene glycol inhibits the loss of cerebral cells after brain injury.

We have tested the effectiveness of polyethylene glycol (PEG) to restore the integrity of neuronal membranes after mechanical damage secondary to severe traumatic brain injury (TBI) produced by a standardized head injury model in rats. We provide additional detail on the standardization of this model, particularly the use and storage of foam bedding that serves to both support the animal during the impact procedure-and as a dampener to the acceleration of the brass weight. Further, we employed a dye exclusion technique using ethidium bromide (EB; quantitative evaluation) and horseradish peroxidase (HRP; qualitative evaluation). Both have been successfully used previously to evaluate neural injury in the spinal cord since they enter cells when their plasma membranes are damaged. We quantified EB labeling (90 microM in 110 microL of sterile saline) after injection into the left lateral ventricle of the rat brain 2 h after injury. At six h after injection and 8 h after injury, the animals were sacrificed and the brains were analyzed. In the injured rat brain, EB entered cells lining and medial to the ventricles, particularly the axons of the corpus callosum. There was minimal EB labeling in uninjured control brains, limited to cells lining the luminal surfaces of the ventricles. Intravenous injections of PEG (1 cc of saline, 30% by volume, 2000 MW) immediately after severe TBI resulted in significantly decreased EB uptake compared with injured control animals. A similar result was achieved using the larger marker, HRP. PEG-treated brains closely resembled those of uninjured animals.