Curcumin as a potential therapeutic agent for treating neurodegenerative diseases.

Neurodegenerative diseases are characterized by the progressive loss of neuronal structure and function, posing a tremendous burden on health systems worldwide. Although the underlying pathological mechanisms for various neurodegenerative diseases are still unclear, a common pathological hallmark is the abundance of neuroinflammatory processes, which affect both disease onset and progression. In this review, we explore the pathways and role of neuroinflammation in various neurodegenerative diseases and further assess the potential use of curcumin, a natural spice with antioxidant and anti-inflammatory properties that has been extensively used worldwide as a traditional medicine and potential therapeutic agent. Following the examination of preclinical and clinical studies that assessed curcumin as a potential therapeutic agent, we highlight the bioavailability of curcumin in the body and discuss both the challenges and benefits of using curcumin as a therapeutic compound for treating neurodegeneration. Although elucidating the involvement of curcumin in aging and neurodegeneration has great potential for developing future CNS-related therapeutic targets, further research is required to elucidate the mechanisms by which Curcumin affects brain physiology, especially BBB integrity, under both physiological and disease conditions.

Chronic neuroinflammation during aging leads to cholinergic neurodegeneration in the mouse medial septum.

BACKGROUND: Low-grade, chronic inflammation in the central nervous system characterized by glial reactivity is one of the major hallmarks for aging-related neurodegenerative diseases like Alzheimer's disease (AD). The basal forebrain cholinergic neurons (BFCN) provide the primary source of cholinergic innervation of the human cerebral cortex and may be differentially vulnerable in various neurodegenerative diseases. However, the impact of chronic neuroinflammation on the cholinergic function is still unclear. METHODS: To gain further insight into age-related cholinergic decline, we investigated the cumulative effects of aging and chronic neuroinflammation on the structure and function of the septal cholinergic neurons in transgenic mice expressing interleukin-6 under the GFAP promoter (GFAP-IL6), which maintains a constant level of gliosis. Immunohistochemistry combined with unbiased stereology, single cell 3D morphology analysis and in vitro whole cell patch-clamp measurements were used to validate the structural and functional changes of BFCN and their microglial environment in the medial septum. RESULTS: Stereological estimation of MS microglia number displayed significant increase across all three age groups, while a significant decrease in cholinergic cell number in the adult and aged groups in GFAP-IL6 mice compared to control. Moreover, we observed age-dependent alterations in the electrophysiological properties of cholinergic neurons and an increased excitability profile in the adult GFAP-IL6 group due to chronic neuroinflammation. These results complimented the significant decrease in hippocampal pyramidal spine density seen with aging and neuroinflammation. CONCLUSIONS: We provide evidence of the significant impact of both aging and chronic glial activation on the cholinergic and microglial numbers and morphology in the MS, and alterations in the passive and active electrophysiological membrane properties of septal cholinergic neurons, resulting in cholinergic dysfunction, as seen in AD. Our results indicate that aging combined with gliosis is sufficient to cause cholinergic disruptions in the brain, as seen in dementias.

Astrocytic K+ clearance during disease progression in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which patients lose motor functions due to progressive loss of motor neurons in the cortex, brainstem, and spinal cord. Whilst the loss of neurons is central to the disease, it is becoming clear that glia, specifically astrocytes, contribute to the onset and progression of neurodegeneration. Astrocytes play an important role in maintaining ion homeostasis in the extracellular milieu and regulate multiple brain functions by altering their extracellular concentrations. In this study, we have investigated the ability of astrocytes to maintain K+ homeostasis in the brain via direct measurement of the astrocytic K+ clearance rate in the motor and somatosensory cortices of an ALS mouse model (SOD1G93A ). Using electrophysiological recordings from acute brain slices, we show region-specific alterations in the K+ clearance rate, which was significantly reduced in the primary motor cortex but not the somatosensory cortex. This decrease was accompanied by significant changes in astrocytic morphology, impaired conductivity via Kir4.1 channels and low coupling ratio in astrocytic networks in the motor cortex, which affected their ability to form the K+ gradient needed to disperse K+ through the astrocytic syncytium. These findings indicate that the supportive function astrocytes typically provide to motoneurons is diminished during disease progression and provides a potential explanation for the increased vulnerability of motoneurons in ALS.

Astrocytic modulation of neuronal signalling.

Neuronal signalling is a key element in neuronal communication and is essential for the proper functioning of the CNS. Astrocytes, the most prominent glia in the brain play a key role in modulating neuronal signalling at the molecular, synaptic, cellular, and network levels. Over the past few decades, our knowledge about astrocytes and their functioning has evolved from considering them as merely a brain glue that provides structural support to neurons, to key communication elements. Astrocytes can regulate the activity of neurons by controlling the concentrations of ions and neurotransmitters in the extracellular milieu, as well as releasing chemicals and gliotransmitters that modulate neuronal activity. The aim of this review is to summarise the main processes through which astrocytes are modulating brain function. We will systematically distinguish between direct and indirect pathways in which astrocytes affect neuronal signalling at all levels. Lastly, we will summarize pathological conditions that arise once these signalling pathways are impaired focusing on neurodegeneration.

Neuromodulation of Astrocytic K+ Clearance.

Potassium homeostasis is fundamental for brain function. Therefore, effective removal of excessive K+ from the synaptic cleft during neuronal activity is paramount. Astrocytes play a key role in K+ clearance from the extracellular milieu using various mechanisms, including uptake via Kir channels and the Na+-K+ ATPase, and spatial buffering through the astrocytic gap-junction coupled network. Recently we showed that alterations in the concentrations of extracellular potassium ([K+]o) or impairments of the astrocytic clearance mechanism affect the resonance and oscillatory behavior of both the individual and networks of neurons. These results indicate that astrocytes have the potential to modulate neuronal network activity, however, the cellular effectors that may affect the astrocytic K+ clearance process are still unknown. In this study, we have investigated the impact of neuromodulators, which are known to mediate changes in network oscillatory behavior, on the astrocytic clearance process. Our results suggest that while some neuromodulators (5-HT; NA) might affect astrocytic spatial buffering via gap-junctions, others (DA; Histamine) primarily affect the uptake mechanism via Kir channels. These results suggest that neuromodulators can affect network oscillatory activity through parallel activation of both neurons and astrocytes, establishing a synergistic mechanism to maximize the synchronous network activity.

Neuromodulation of Glial Function During Neurodegeneration.

Glia, a non-excitable cell type once considered merely as the connective tissue between neurons, is nowadays acknowledged for its essential contribution to multiple physiological processes including learning, memory formation, excitability, synaptic plasticity, ion homeostasis, and energy metabolism. Moreover, as glia are key players in the brain immune system and provide structural and nutritional support for neurons, they are intimately involved in multiple neurological disorders. Recent advances have demonstrated that glial cells, specifically microglia and astroglia, are involved in several neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Epilepsy, Parkinson's disease (PD), Alzheimer's disease (AD), and frontotemporal dementia (FTD). While there is compelling evidence for glial modulation of synaptic formation and regulation that affect neuronal signal processing and activity, in this manuscript we will review recent findings on neuronal activity that affect glial function, specifically during neurodegenerative disorders. We will discuss the nature of each glial malfunction, its specificity to each disorder, overall contribution to the disease progression and assess its potential as a future therapeutic target.

Method for Prolonged Incubation of Brain Slices.

Slices of neuronal tissue maintain a high degree of topographical and functional properties of neurons and glia and therefore are extensively used for measurements of neuronal activity at the molecular, cellular and network levels. However, the lifespan of slice preparations is narrow, averaging of 6-8 hours. Moreover, the average viability of brain slices varies according to animal age and region of interest, leading to the high variability and low reproducibility of recorded data. Previous techniques to increase the viability of brain slices focused on reducing cytotoxicity by chemical means, including alterations of the artificial cerebrospinal fluid (aCSF) composition to alleviate the direct damage of the slicing procedure or adding protective antioxidants to reduce cellular deterioration. In this protocol, we use a combination of hypothermia with firm control of the aCSF conditions in the recovery chamber (pH, temperature, and bacteria levels) to extend the slice viability significantly. Given the breadth of its usage, improving slice viability and longevity can considerably increase data reproducibility and reduce the cost, time, and number of animals used in neurophysiological studies.

Generating Brain Waves, the Power of Astrocytes.

Synchronization of neuronal activity in the brain underlies the emergence of neuronal oscillations termed "brain waves", which serve various physiological functions and correlate with different behavioral states. It has been postulated that at least ten distinct mechanisms are involved in the formulation of these brain waves, including variations in the concentration of extracellular neurotransmitters and ions, as well as changes in cellular excitability. In this mini review we highlight the contribution of astrocytes, a subtype of glia, in the formation and modulation of brain waves mainly due to their close association with synapses that allows their bidirectional interaction with neurons, and their syncytium-like activity via gap junctions that facilitate communication to distal brain regions through Ca2+ waves. These capabilities allow astrocytes to regulate neuronal excitability via glutamate uptake, gliotransmission and tight control of the extracellular K+ levels via a process termed K+ clearance. Spatio-temporal synchrony of activity across neuronal and astrocytic networks, both locally and distributed across cortical regions, underpins brain states and thereby behavioral states, and it is becoming apparent that astrocytes play an important role in the development and maintenance of neural activity underlying these complex behavioral states.

The differential impact of acute microglia activation on the excitability of cholinergic neurons in the mouse medial septum.

The medial septal nucleus is one of the basal forebrain nuclei that projects cholinergic input to the hippocampus and cortex. Two of the hallmarks of Alzheimer's disease (AD) are a significant loss of cholinergic transmission and neuroinflammation, and it has been suggested that these two hallmarks are causally linked to the medial septum. Therefore, we have investigated the age-related susceptibility of medial septal cholinergic neurons to glial activation, mediated via peripheral administration of lipopolysaccharide (500 µg/kg) into ChAT(BAC)-eGFP mice at different ages (3-22 months). Our results show that during normal aging, cholinergic neurons experience a bi-phasic excitability profile, in which increased excitability at adulthood (ages ranging between 9 and 12 months) decreases in aged animals (> 18 months). Moreover, activation of glia had a differential impact on mice from different age groups, affecting K+ conductances in young and adult animals, without affecting aged mice. These findings provide a potential explanation for the increased vulnerability of cholinergic neurons to neuroinflammation with aging as reported previously, thus providing a link to the impact of acute neuroinflammation in AD.

Dynamic interplay between H-current and M-current controls motoneuron hyperexcitability in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease (MND) in which humans lose motor functions due to progressive loss of motoneurons in the cortex, brainstem, and spinal cord. In patients and in animal models of MND it has been observed that there is a change in the properties of motoneurons, termed neuronal hyperexcitability, which is an exaggerated response of the neurons to a stimulus. Previous studies suggested neuronal excitability is one of the leading causes for neuronal loss, however the factors that instigate excitability in neurons over the course of disease onset and progression are not well understood, as these studies have looked mainly at embryonic or early postnatal stages (pre-symptomatic). As hyperexcitability is not a static phenomenon, the aim of this study was to assess the overall excitability of upper motoneurons during disease progression, specifically focusing on their oscillatory behavior and capabilities to fire repetitively. Our results suggest that increases in the intrinsic excitability of motoneurons are a global phenomenon of aging, however the cellular mechanisms that underlie this hyperexcitability are distinct in SOD1G93A ALS mice compared with wild-type controls. The ionic mechanism driving increased excitability involves alterations of the expression levels of HCN and KCNQ channel genes leading to a complex dynamic of H-current and M-current activation. Moreover, we show a negative correlation between the disease onset and disease progression, which correlates with a decrease in the expression level of HCN and KCNQ channels. These findings provide a potential explanation for the increased vulnerability of motoneurons to ALS with aging.

Astrocytic modulation of cortical oscillations.

Brain waves are rhythmic voltage oscillations emerging from the synchronization of individual neurons into a neuronal network. These oscillations range from slow to fast fluctuations, and are classified by power and frequency band, with different frequency bands being associated with specific behaviours. It has been postulated that at least ten distinct mechanisms are required to cover the frequency range of neural oscillations, however the mechanisms that gear the transition between distinct oscillatory frequencies are unknown. In this study, we have used electrophysiological recordings to explore the involvement of astrocytic K+ clearance processes in modulating neural oscillations at both network and cellular levels. Our results indicate that impairment of astrocytic K+ clearance capabilities, either through blockade of K+ uptake or astrocytic connectivity, enhance network excitability and form high power network oscillations over a wide range of frequencies. At the cellular level, local increases in extracellular K+ results in modulation of the oscillatory behaviour of individual neurons, which underlies the network behaviour. Since astrocytes are central for maintaining K+ homeostasis, our study suggests that modulation of their inherent capabilities to clear K+ from the extracellular milieu is a potential mechanism to optimise neural resonance behaviour and thus tune neural oscillations.

Impairments in Motor Neurons, Interneurons and Astrocytes Contribute to Hyperexcitability in ALS: Underlying Mechanisms and Paths to Therapy.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of motor neurons leading to progressive paralysis and death. Using transcranial magnetic stimulation (TMS) and nerve excitability tests, several clinical studies have identified that cortical and peripheral hyperexcitability are among the earliest pathologies observed in ALS patients. The changes in the electrophysiological properties of motor neurons have been identified in both sporadic and familial ALS patients, despite the diverse etiology of the disease. The mechanisms behind the change in neuronal signalling are not well understood, though current findings implicate intrinsic changes in motor neurons and dysfunction of cells critical in regulating motor neuronal excitability, such as astrocytes and interneurons. Alterations in ion channel expression and/or function in motor neurons has been associated with changes in cortical and peripheral nerve excitability. In addition to these intrinsic changes in motor neurons, inhibitory signalling through GABAergic interneurons is also impaired in ALS, likely contributing to increased neuronal excitability. Astrocytes have also recently been implicated in increasing neuronal excitability in ALS by failing to adequately regulate glutamate levels and extracellular K+ concentration at the synaptic cleft. As hyperexcitability is a common and early feature of ALS, it offers a therapeutic and diagnostic target. Thus, understanding the underlying pathways and mechanisms leading to hyperexcitability in ALS offers crucial insight for future development of ALS treatments.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

Astrocytic modulation of neuronal excitability through K+ spatial buffering.

The human brain contains two major cell populations, neurons and glia. While neurons are electrically excitable and capable of discharging short voltage pulses known as action potentials, glial cells are not. However, astrocytes, the prevailing subtype of glia in the cortex, are highly connected and can modulate the excitability of neurons by changing the concentration of potassium ions in the extracellular environment, a process called K+ clearance. During the past decade, astrocytes have been the focus of much research, mainly due to their close association with synapses and their modulatory impact on neuronal activity. It has been shown that astrocytes play an essential role in normal brain function including: nitrosative regulation of synaptic release in the neocortex, synaptogenesis, synaptic transmission and plasticity. Here, we discuss the role of astrocytes in network modulation through their K+ clearance capabilities, a theory that was first raised 50 years ago by Orkand and Kuffler. We will discuss the functional alterations in astrocytic activity that leads to aberrant modulation of network oscillations and synchronous activity.

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (>24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 °C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at >24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Differential effect of brief electrical stimulation on voltage-gated potassium channels.

Electrical stimulation of neuronal tissue is a promising strategy to treat a variety of neurological disorders. The mechanism of neuronal activation by external electrical stimulation is governed by voltage-gated ion channels. This stimulus, typically brief in nature, leads to membrane potential depolarization, which increases ion flow across the membrane by increasing the open probability of these voltage-gated channels. In spiking neurons, it is activation of voltage-gated sodium channels (NaV channels) that leads to action potential generation. However, several other types of voltage-gated channels are expressed that also respond to electrical stimulation. In this study, we examine the response of voltage-gated potassium channels (KV channels) to brief electrical stimulation by whole cell patch-clamp electrophysiology and computational modeling. We show that nonspiking amacrine neurons of the retina exhibit a large variety of responses to stimulation, driven by different KV-channel subtypes. Computational modeling reveals substantial differences in the response of specific KV-channel subtypes that is dependent on channel kinetics. This suggests that the expression levels of different KV-channel subtypes in retinal neurons are a crucial predictor of the response that can be obtained. These data expand our knowledge of the mechanisms of neuronal activation and suggest that KV-channel expression is an important determinant of the sensitivity of neurons to electrical stimulation.NEW & NOTEWORTHY This paper describes the response of various voltage-gated potassium channels (KV channels) to brief electrical stimulation, such as is applied during prosthetic electrical stimulation. We show that the pattern of response greatly varies between KV channel subtypes depending on activation and inactivation kinetics of each channel. Our data suggest that problems encountered when artificially stimulating neurons such as cessation in firing at high frequencies, or "fading," may be attributed to KV-channel activation.

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue.

Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45-150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6-8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 µM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and ß) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.