Extracellular S100B inhibits A-type voltage-gated potassium currents and increases L-type voltage-gated calcium channel activity in dopaminergic neurons.

Parkinson's disease (PD) is associated with an increase in secreted S100B within the midbrain and cerebrospinal fluid. In addition, S100B overexpression in mice accelerates the loss of substantia nigra pars compacta dopaminergic (DA) neurons, suggesting a role for this protein in PD pathogenesis. We found that in the mouse SNc, S100B labeled astrocytic processes completely envelop the somata of tyrosine hydroxylase (TH) expressing DA neurons only in male mice. These data suggest that an increase in S100B secretion by astrocytes within the midbrain could play a role in DA dysfunction during early PD. We therefore asked if acute exposure to extracellular S100B alters the activity of identified TH expressing DA neurons in primary mouse midbrain cultures. Acute exposure to 50 pM S100B specifically inhibited A-type voltage-gated potassium currents in TH+ , but not TH- neurons. This was accompanied by ~2-fold increases in the frequency of both intrinsic firing, as well as L-type voltage-gated calcium channel (VGCC)-mediated calcium fluxes only in TH+ neurons. Further, exposure to 100 µM 4-aminopyridine (4-AP), an A-type voltage-gated potassium channel inhibitor, mimicked the S100B mediated increase in intrinsic firing and L-type VGCC-mediated calcium fluxes in TH+ neurons. Taken together, our finding that extracellular S100B alters the activity of native DA neurons via an inhibition of A-type voltage-gated potassium channels has important implications for understanding the pathophysiology of early PD.

Cytisine is neuroprotective in female but not male 6-hydroxydopamine lesioned parkinsonian mice and acts in combination with 17-ß-estradiol to inhibit apoptotic endoplasmic reticulum stress in dopaminergic neurons.

Apoptotic endoplasmic reticulum (ER) stress is a major mechanism for dopaminergic (DA) loss in Parkinson's disease (PD). We assessed if low doses of the partial α4ß2 nicotinic acetylcholine receptor agonist, cytisine attenuates apoptotic ER stress and exerts neuroprotection in substantia nigra pars compacta (SNc) DA neurons. Alternate day intraperitoneal injections of 0.2 mg/kg cytisine were administered to female and male mice with 6-hydroxydopamine (6-OHDA) lesions in the dorsolateral striatum, which caused unilateral degeneration of SNc DA neurons. Cytisine attenuated 6-OHDA-induced PD-related behaviors in female, but not in male mice. We also found significant reductions in tyrosine hydroxylase (TH) loss within the lesioned SNc of female, but not male mice. In contrast to female mice, DA neurons within the lesioned SNc of male mice showed a cytisine-induced pathological increase in the nuclear translocation of the pro-apoptotic ER stress protein, C/EBP homologous protein (CHOP). To assess the role of estrogen in cytisine neuroprotection in female mice, we exposed primary mouse DA cultures to either 10 nM 17-ß-estradiol and 200 nM cytisine or 10 nM 17-ß-estradiol alone. 17-ß-estradiol reduced expression of CHOP, whereas cytisine exposure reduced 6-OHDA-mediated nuclear translocation of two other ER stress proteins, activating transcription factor 6 and x-box-binding protein 1, but not CHOP. Taken together, these data show that cytisine and 17-ß-estradiol work in combination to inhibit all three arms (activating transcription factor 6, x-box-binding protein 1, and CHOP) of apoptotic ER stress signaling in DA neurons, which can explain the neuroprotective effect of low-dose cytisine in female mice.

Macrophage Migration Inhibitory Factor Alters Functional Properties of CA1 Hippocampal Neurons in Mouse Brain Slices.

Neuroinflammation is implicated in a host of neurological insults, such as traumatic brain injury (TBI), ischemic stroke, Alzheimer's disease, Parkinson's disease, and epilepsy. The immune response to central nervous system (CNS) injury involves sequelae including the release of numerous cytokines and chemokines. Macrophage migration inhibitory factor (MIF), is one such cytokine that is elevated following CNS injury, and is associated with the prognosis of TBI, and ischemic stroke. MIF has been identified in astrocytes and neurons, and some of the trophic actions of MIF have been related to its direct and indirect actions on astrocytes. However, the potential modulation of CNS neuronal function by MIF has not yet been explored. This study tests the hypothesis that MIF can directly influence hippocampal neuronal function. MIF was microinjected into the hippocampus and the genetically encoded calcium indicator, GCaMP6f, was used to measure Ca2+ events in acute adult mouse brain hippocampal slices. Results demonstrated that a single injection of 200 ng MIF into the hippocampus significantly increased baseline calcium signals in CA1 pyramidal neuron somata, and altered calcium responses to N-methyl-d-aspartate (NMDA) + D-serine in pyramidal cell apical dendrites located in the stratum radiatum. These data are the first to show direct effects of MIF on hippocampal neurons and on NMDA receptor function. Considering that MIF is elevated after brain insults such as TBI, the data suggest that, in addition to the previously described role of MIF in astrocyte reactivity, elevated MIF can have significant effects on neuronal function in the hippocampus.

Intracranial pressure monitoring in diffuse brain injury-why the developing world needs it more?

BACKGROUND: Use of ICP monitoring is considered to be part of "standard of care" in management of severe traumatic brain injury, but it is rarely used in developing countries. The authors present a study which evaluates the efficacy and outcomes of ICP monitoring at a high-volume trauma center in India. METHODS: Data on management and outcomes for 126 patients who were admitted with diffuse traumatic brain injury (GCS 3-8) were studied prospectively over an 18-month period. These patients were treated by one of the two specific protocols: ICP monitoring-based or non-ICP monitoring-based. The primary outcome was measured based on 2 weeks mortality and GOS-E at 1, 3, and 6 months. Secondary outcome was measured based on need for brain-specific treatment, length of ICU stay, and radiation exposure. RESULTS: Mortality in a subset of patients who underwent surgical intervention later due to increased ICP values, drop in GCS, or radiological deterioration was noted to be significantly lower in the ICP monitoring group (p = 0.03), in spite of statistically insignificant difference in overall mortality rates between groups. GOS-E scores at 1 month were significantly better (p = 0.033) in ICP monitoring group, even though they equalized at 3 and 6 months. The need for brain-specific treatment (p < 0.001), radiation exposure (p < 0.001), and length of ICU stay (p = 0.013) was significantly lower in the ICP monitoring group. CONCLUSIONS: ICP monitoring-based treatment protocol helps in achieving faster recovery; lowers mortality rates in operated patients; and reduces ICU stay, radiation exposure, and the need for brain-specific treatment.

Smoking-Relevant Nicotine Concentration Attenuates the Unfolded Protein Response in Dopaminergic Neurons.

Retrospective epidemiological studies show an inverse correlation between susceptibility to Parkinson's disease and a person's history of tobacco use. Animal model studies suggest nicotine as a neuroprotective agent and nicotinic acetylcholine (ACh) receptors (nAChRs) as targets for neuroprotection, but the underlying neuroprotective mechanism(s) are unknown. We cultured mouse ventral midbrain neurons for 3 weeks. Ten to 20% of neurons were dopaminergic (DA), revealed by tyrosine hydroxylase (TH) immunoreactivity. We evoked mild endoplasmic reticulum (ER) stress with tunicamycin (Tu), producing modest increases in the level of nuclear ATF6, phosphorylated eukaryotic initiation factor 2α, nuclear XBP1, and the downstream proapoptotic effector nuclear C/EBP homologous protein. We incubated cultures for 2 weeks with 200 nm nicotine, the approximate steady-state concentration between cigarette smoking or vaping, or during nicotine patch use. Nicotine incubation suppressed Tu-induced ER stress and the unfolded protein response (UPR). Study of mice with fluorescent nAChR subunits showed that the cultured TH+ neurons displayed α4, α6, and ß3 nAChR subunit expression and ACh-evoked currents. Gene expression profile in cultures from TH-eGFP mice showed that the TH+ neurons also express several other genes associated with DA release. Nicotine also upregulated ACh-induced currents in DA neurons by ∼2.5-fold. Thus, nicotine, at a concentration too low to activate an appreciable fraction of plasma membrane nAChRs, induces two sequelae of pharmacological chaperoning in the ER: UPR suppression and nAChR upregulation. Therefore, one mechanism of neuroprotection by nicotine is pharmacological chaperoning, leading to UPR suppression. Measuring this pathway may help in assessing neuroprotection. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) cannot yet be cured or prevented. However, many retrospective epidemiological studies reveal that PD is diagnosed less frequently in tobacco users. Existing programs attempting to develop nicotinic drugs that might exert this apparent neuroprotective effect are asking whether agonists, antagonists, partial agonists, or channel blockers show the most promise. The underlying logic resembles the previous development of varenicline for smoking cessation. We studied whether, and how, nicotine produces neuroprotective effects in cultured dopaminergic neurons, an experimentally tractable, mechanistically revealing neuronal system. We show that nicotine, operating via nicotinic receptors, does protect these neurons against endoplasmic reticulum stress. However, the mechanism is probably "inside-out": pharmacological chaperoning in the endoplasmic reticulum. This cellular-level insight could help to guide neuroprotective strategies.

Pharmacological chaperoning of nAChRs: a therapeutic target for Parkinson's disease.

Chronic exposure to nicotine results in an upregulation of neuronal nicotinic acetylcholine receptors (nAChRs) at the cellular plasma membrane. nAChR upregulation occurs via nicotine-mediated pharmacological receptor chaperoning and is thought to contribute to the addictive properties of tobacco as well as relapse following smoking cessation. At the subcellular level, pharmacological chaperoning by nicotine and nicotinic ligands causes profound changes in the structure and function of the endoplasmic reticulum (ER), ER exit sites, the Golgi apparatus and secretory vesicles of cells. Chaperoning-induced changes in cell physiology exert an overall inhibitory effect on the ER stress/unfolded protein response. Cell autonomous factors such as the repertoire of nAChR subtypes expressed by neurons and the pharmacological properties of nicotinic ligands (full or partial agonist versus competitive antagonist) govern the efficiency of receptor chaperoning and upregulation. Together, these findings are beginning to pave the way for developing pharmacological chaperones to treat Parkinson's disease and nicotine addiction.

Heterogeneous brain region-specific responses to astrocytic mitochondrial DNA damage in mice.

Astrocytes use Ca 2+ signals to regulate multiple aspects of normal and pathological brain function. Astrocytes display context-specific diversity in their functions, and in their response to noxious stimuli between brain regions. Indeed, astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on their ATP generation and Ca 2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca 2+ signaling in astrocytes, the extent of this regulation into the rich diversity of astrocytes in different brain regions remains largely unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for proper astrocyte function, however, few insights into possible diverse responses to this noxious stimulus from astrocytes in different brain areas were reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI, which expresses the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two distinct brain regions, the dorsolateral striatum, and the hippocampal dentate gyrus, and we show that Mito-PstI can induce astrocytic mtDNA loss in vivo , but with remarkable brain-region-dependent differences on mitochondrial dynamics, spontaneous Ca 2+ fluxes and astrocytic as well as microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca 2+ signaling in a brain-region-selective manner.

Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures.

We exploit the optical and spatial features of subwavelength nanostructures to examine individual receptors on the plasma membrane of living cells. Receptors were sequestered in portions of the membrane projected into zero-mode waveguides. Using single-step photobleaching of green fluorescent protein incorporated into individual subunits, the resulting spatial isolation was used to measure subunit stoichiometry in α4ß4 and α4ß2 nicotinic acetylcholine and P2X2 ATP receptors. We also show that nicotine and cytisine have differential effects on α4ß2 stoichiometry.

Astrocytes and Alpha-Synuclein: Friend or Foe?

Despite its devastating disease burden and alarming prevalence, the etiology of Parkinson's disease (PD) remains to be completely elucidated. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta and this correlates with the accumulation of misfolded α-synuclein. While the aggregation of α-synuclein in the form of Lewy bodies or Lewy neurites is a well-established intraneuronal hallmark of the disease process, our understanding of the glial contribution to aberrant α-synuclein proteostasis is lacking. In this regard, restoring astrocyte function during early PD could offer a promising therapeutic avenue and understanding the involvement of astrocytes in handling/mishandling of α-synuclein is of particular interest. Here, we explore the growing body of scientific literature implicating aberrant astrocytic α-synuclein proteostasis with the seemingly inexorable pathological sequelae typifying PD. We also provide a perspective on how heterogeneity in the morphological relationship between astrocytes and neurons will need to be considered in the context of PD pathogenesis.

Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum.

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases the risk for neurodegeneration. Among various cells that participate in BBB and NVU function, calcium signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with altered calcium signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed GCaMP6f in DLS astrocytes of young (3-4 months), middle-aged (12-15 months) and aging (20-30 months) mice. Compared to endfeet in young mice, DLS endfeet in aging mice demonstrated decreased calreticulin expression, and alterations to both spontaneous membrane-associated and mitochondrial calcium signals. While young mice required both extracellular and endoplasmic reticulum calcium sources for endfoot signals, middle-aged and aging mice showed heavy dependence on endoplasmic reticulum calcium. Thus, astrocytic endfeet show significant changes in calcium buffering and sources throughout the lifespan, which is important for understanding mechanisms by which aging impairs the BBB and NVU.

Pharmacological chaperoning of nicotinic acetylcholine receptors reduces the endoplasmic reticulum stress response.

We report the first observation that endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) can decrease when a central nervous system drug acts as an intracellular pharmacological chaperone for its classic receptor. Transient expression of α4ß2 nicotinic receptors (nAChRs) in Neuro-2a cells induced the nuclear translocation of activating transcription factor 6 (ATF6), which is part of the UPR. Cells were exposed for 48 h to the full agonist nicotine, the partial agonist cytisine, or the competitive antagonist dihydro-ß-erythroidine; we also tested mutant nAChRs that readily exit the ER. Each of these four manipulations increased Sec24D-enhanced green fluorescent protein fluorescence of condensed ER exit sites and attenuated translocation of ATF6-enhanced green fluorescent protein to the nucleus. However, we found no correlation among the manipulations regarding other tested parameters [i.e., changes in nAChR stoichiometry (α4(2)ß2(3) versus α4(3)ß2(2)), changes in ER and trans-Golgi structures, or the degree of nAChR up-regulation at the plasma membrane]. The four manipulations activated 0 to 0.4% of nAChRs, which shows that activation of the nAChR channel did not underlie the reduced ER stress. Nicotine also attenuated endogenously expressed ATF6 translocation and phosphorylation of eukaryotic initiation factor 2α in mouse cortical neurons transfected with α4ß2 nAChRs. We conclude that, when nicotine accelerates ER export of α4ß2 nAChRs, this suppresses ER stress and the UPR. Suppression of a sustained UPR may explain the apparent neuroprotective effect that causes the inverse correlation between a person's history of tobacco use and susceptibility to developing Parkinson's disease. This suggests a novel mechanism for neuroprotection by nicotine.

α7ß2 nicotinic acetylcholine receptors assemble, function, and are activated primarily via their α7-α7 interfaces.

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and ß2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and ß2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and ß2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and ß2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7ß2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7ß2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7ß2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-ß-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-ß2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7ß2 nAChR current amplitudes and challenges in identifying the function of native α7ß2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of ß2 subunits within the α7ß2 nAChRs.

Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine.

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4ß4 nAChRs, but many α4ß2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the ß4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4ß4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2-3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4ß4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 µM) did not alter the PM insertion frequency or trafficking behavior of α4ß4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4ß2-containing vesicle fusions at the PM; this stage in α4ß2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.

Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in cys-loop receptors, exemplified by nicotinic α4ß2.

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and ß2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)(3)(ß2)(2)vs. (α4)(2)(ß2)(3). Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)(3)(ß2)(2) exceeds that of (α4)(2)(ß2)(3). The simulations account for published data on transfected Neuro2a cells in which α4ß2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)(3)(ß2)(2) stoichiometry to mostly (α4)(2)(ß2)(3). The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)(2)(ß2)(3) and (α4)(3)(ß2)(2) populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.

Microneurosurgery for Spinal Dural Arteriovenous Fistula- Operative Video.

Background and Introduction: Spinal dural arteriovenous fistula (SDAVF) is a rare but curable condition. Microsurgery is a highly effective and readily affordable treatment modality. Objective: We present a surgical video of SDAVF to demonstrate the operative nuances involved. Surgical Technique: A 53-year-old wheelchair-bound man with spastic paraparesis for 1.5 years was found to have a SDAVF at L1/2 level with a single fistula point. During surgery, a L1-L2 laminectomy and durotomy revealed a dilated vein accompanying the nerve root exiting L1/2 foramen that showed early filling on indocyanine green (ICG) video angiography. This vein was occluded, and a segment of this vein was removed during surgery, which led to resumption of normal spinal cord perfusion. Results: The patient showed gradual recovery of lower limb motor power and improved to assisted ambulation after 3 months. Conclusions: Surgery is a simple, effective, and cost-effective treatment option in SDAVF.

Functional Assessment of Stroke-Induced Regulation of miR-20a-3p and Its Role as a Neuroprotectant.

MicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17-92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.

Calcium signals in astrocytes of the fly brain promote sleep.

Astrocytes are morphologically and functionally linked to neuronal synapses, and can regulate the activity of neural circuits, brain function, and behavior. However, the molecular mechanisms by which astrocytes regulate fundamental biological processes such as sleep are not completely understood. Wu and colleagues show that an increase in calcium signals within the processes of astrocytes of the fruit fly brain can promote sleep by upregulating the expression of a monoamine receptor, TyrRII, which in turn activates sleep promoting neurons via the astrocytic release of an interleukin-1 analog, spatzle. This study provides compelling evidence for a novel molecular mechanism by which increases in astrocytic calcium signals can induce sleep by activating sleep promoting neurons in the fly brain.

Adeno-Associated Virus Expression of α-Synuclein as a Tool to Model Parkinson's Disease: Current Understanding and Knowledge Gaps.

Parkinson's disease (PD) is the second most common neurodegenerative disorder in the aging population and is characterized by a constellation of motor and non-motor symptoms. The abnormal aggregation and spread of alpha-synuclein (α-syn) is thought to underlie the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc), leading to the development of PD. It is in this context that the use of adeno-associated viruses (AAVs) to express a-syn in the rodent midbrain has become a popular tool to model SNc DA neuron loss during PD. In this review, we summarize results from two decades of experiments using AAV-mediated a-syn expression in rodents to model PD. Specifically, we outline aspects of AAV vectors that are particularly relevant to modeling a-syn dysfunction in rodent models of PD such as changes in striatal neurochemistry, a-syn biochemistry, and PD-related behaviors resulting from AAV-mediated a-syn expression in the midbrain. Finally, we discuss the emerging role of astrocytes in propagating a-syn pathology, and point to future directions for employing AAVs as a tool to better understand how astrocytes contribute to a-syn pathology during the development of PD. We envision that lessons learned from two decades of utilizing AAVs to express a-syn in the rodent brain will enable us to develop an optimized set of parameters for gaining a better understanding of how a-syn leads to the development of PD.

Emerging Roles for Aberrant Astrocytic Calcium Signals in Parkinson's Disease.

Astrocytes display a plethora of spontaneous Ca2+ signals that modulate vital functions of the central nervous system (CNS). This suggests that astrocytic Ca2+ signals also contribute to pathological processes in the CNS. In this context, the molecular mechanisms by which aberrant astrocytic Ca2+ signals trigger dopaminergic neuron loss during Parkinson's disease (PD) are only beginning to emerge. Here, we provide an evidence-based perspective on potential mechanisms by which aberrant astrocytic Ca2+ signals can trigger dysfunction in three distinct compartments of the brain, viz., neurons, microglia, and the blood brain barrier, thereby leading to PD. We envision that the coming decades will unravel novel mechanisms by which aberrant astrocytic Ca2+ signals contribute to PD and other neurodegenerative processes in the CNS.

Astrocytic mitochondria in adult mouse brain slices show spontaneous calcium influx events with unique properties.

Astrocytes govern critical aspects of brain function via spontaneous calcium signals in their soma and processes. A significant proportion of these spontaneous astrocytic calcium events are associated with mitochondria, however, the extent, sources, or kinetics of astrocytic mitochondrial calcium influx have not been studied in the adult mouse brain. To measure calcium influx into astrocytic mitochondria in situ, we generated an adeno-associated virus (AAV) with the astrocyte-specific GfaABC1D promoter driving expression of the genetically encoded calcium indicator, GCaMP6f tagged to mito7, a mitochondrial matrix targeted signal sequence. Using this construct, we observed AAV-mediated expression of GCaMP6f in adult mouse astrocytic mitochondria that co-localized with MitoTracker deep red (MTDR) in the dorsolateral striatum (DLS) and in the hippocampal stratum radiatum (HPC). Astrocytic mitochondria co-labeled with MTDR and GCaMP6f displayed robust, spontaneous calcium influx events in situ, with subcellular differences in calcium influx kinetics between somatic, branch, and branchlet mitochondria, and inter-regional differences between mitochondria in DLS and HPC astrocytes. Calcium influx into astrocytic mitochondria was strongly dependent on endoplasmic reticulum calcium stores, but did not require the mitochondrial calcium uniporter, MCU. Exposure to either glutamate, D1 or D2 dopamine receptor agonists increased calcium influx in some mitochondria, while simultaneously decreasing calcium influx in other mitochondria from the same astrocyte. These findings show that astrocytic mitochondria possess unique properties with regard to their subcellular morphology, mechanisms of calcium influx, and responses to neurotransmitter receptor agonists. Our results have important implications for understanding the role of astrocytic mitochondria during pathological processes.