Increased neuronal activity in motor cortex reveals prominent calcium dyshomeostasis in tauopathy mice.

Perturbed neuronal Ca2+ homeostasis is implicated in Alzheimer's disease, which has primarily been demonstrated in mice with amyloid-ß deposits but to a lesser and more variable extent in tauopathy models. In this study, we injected AAV to express Ca2+ indicator in layer II/III motor cortex neurons and measured neuronal Ca2+ activity by two photon imaging in awake transgenic JNPL3 tauopathy and wild-type mice. Various biochemical measurements were conducted in postmortem mouse brains for mechanistic insight and a group of animals received two intravenous injections of a tau monoclonal antibody spaced by four days to test whether the Ca2+ dyshomeostasis was related to pathological tau protein. Under running conditions, we found abnormal neuronal Ca2+ activity in tauopathy mice compared to age-matched wild-type mice with higher frequency of Ca2+ transients, lower amplitude of peak Ca2+ transients and lower total Ca2+ activity in layer II/III motor cortex neurons. While at resting conditions, only Ca2+ frequency was increased. Brain levels of soluble pathological tau correlated better than insoluble tau levels with the degree of Ca2+ dysfunction in tauopathy mice. Furthermore, tau monoclonal antibody 4E6 partially rescued Ca2+ activity abnormalities in tauopathy mice after two intravenous injections and decreased soluble pathological tau protein within the brain. This correlation and antibody effects strongly suggest that the neuronal Ca2+ dyshomeostasis is causally linked to pathological tau protein. These findings also reveal more pronounced neuronal Ca2+ dysregulation in tauopathy mice than previously reported by two-photon imaging that can be partially corrected with an acute tau antibody treatment.

Depletion of astrocytic transglutaminase 2 improves injury outcomes.

Astrocytes play an indispensable role in maintaining a healthy, functional neural network in the central nervous system (CNS). A primary function of CNS astrocytes is to support the survival and function of neurons. In response to injury, astrocytes take on a reactive phenotype, which alters their molecular functions. Reactive astrocytes have been reported to be both beneficial and harmful to the CNS recovery process subsequent to injury. Understanding the molecular processes and regulatory proteins that determine the extent to which an astrocyte hinders or supports neuronal survival is important within the context of CNS repair. One protein that plays a role in modulating cellular survival is transglutaminase 2 (TG2). Global deletion of TG2 results in beneficial outcomes subsequent to in vivo ischemic brain injury. Ex vivo studies have also implicated TG2 as a negative regulator of astrocyte viability subsequent to injury. In this study we show that knocking down TG2 in astrocytes significantly increases their ability to protect neurons from oxygen glucose deprivation (OGD)/reperfusion injury. To begin to understand how deletion of TG2 in astrocytes improves their ability to protect neurons from injury, we performed transcriptome analysis of wild type and TG2-/- astrocytes. TG2 deletion resulted in alterations in genes involved in extracellular matrix remodeling, cell adhesion and axon growth/guidance. In addition, the majority of genes that showed increases in the TG2-/- astrocytes had predicted cJun/AP-1 binding motifs in their promoters. Furthermore, phospho-cJun levels were robustly elevated in TG2-/- astrocytes, a finding which was consistent with the increase in expression of AP-1 responsive genes. These in vitro data were subsequently extended into an in vivo model to determine whether the absence of astrocytic TG2 improves outcomes after CNS injury. Our results show that, following a spinal cord injury, scar formation is significantly attenuated in mice with astrocyte-specific TG2 deletion compared to mice expressing normal TG2 levels. Taken together, these data indicate that TG2 plays a pivotal role in mediating reactive astrocyte properties following CNS injury. Further, the data suggest that limiting the AP-1 mediated pro-survival injury response may be a contributing factor to that the detrimental effects of astrocytic TG2.

Nuclear transglutaminase 2 directly regulates expression of cathepsin S in rat cortical neurons.

Transglutaminase 2 (TG2) is a protein that modulates neuronal survival processes. Although TG2 is primarily cytosolic, data have suggested the nuclear localization of TG2 is strongly associated with neuronal viability. Depletion of TG2 in neurons results in neurite retraction and loss of viability, which is likely due to a dysregulation in gene expression. To begin to understand how TG2 regulates neuronal gene expression, chromatin immunoprecipitation was performed in neurons with TG2 overexpression. The resulting genomic DNA was recovered and sequenced. Bioinformatics analyses revealed that a signature DNA motif was enriched in the TG2 immunoprecipitated genomic DNA. In particular, this motif strongly mapped to a region proximate to the gene Ctss (cathepsin S). Knockdown of TG2 resulted in a significant increase in cathepsin S expression, which preceded the loss of neuronal viability. This is the first demonstration that TG2 directly associates with genomic DNA and regulates gene expression in neurons. Given that expression of cathepsin S is increased in neurological disease states, our data suggest that TG2 may play a role in promoting neuron health in part by repressing the expression of cathepsin S. Overall these data provide new insights into the function of nuclear TG2 in neurons.

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons.

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.

Inhibition or ablation of transglutaminase 2 impairs astrocyte migration.

Astrocytes play numerous complex roles that support and facilitate the function of neurons. Further, when there is an injury to the central nervous system (CNS) they can both facilitate or ameliorate functional recovery depending on the location and severity of the injury. When a CNS injury is relatively severe a glial scar is formed, which is primarily composed of astrocytes. The glial scar can be both beneficial, by limiting inflammation, and detrimental, by preventing neuronal projections, to functional recovery. Thus, understanding the processes and proteins that regulate astrocyte migration in response to injury is still of fundamental importance. One protein that is likely involved in astrocyte migration is transglutaminase 2 (TG2); a multifunctional protein expressed ubiquitously throughout the brain. Its functions include transamidation and GTPase activity, among others, and previous studies have implicated TG2 as a regulator of migration. Therefore, we examined the role of TG2 in primary astrocyte migration subsequent to injury. Using wild type or TG2-/- astrocytes, we manipulated the different functions and conformation of TG2 with novel irreversible inhibitors or mutant versions of the protein. Results showed that both inhibition and ablation of TG2 in primary astrocytes significantly inhibit migration. Additionally, we show that the deficiency in migration caused by deletion of TG2 can only be rescued with the native protein and not with mutants. Finally, the addition of TGFß rescued the migration deficiency independent of TG2. Taken together, our study shows that transamidation and GTP/GDP-binding are necessary for inhibiting astrocyte migration and it is TGFß independent.

Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons.

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma and processes, implying a crucial role in iron accumulation from NTBI and transferrin-bound iron (TBI) by hippocampal neurons.

Single-domain antibody-based protein degrader for synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Single-Domain Antibody-Based Protein Degrader for Synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Neuronal hypofunction and network dysfunction in a mouse model at an early stage of tauopathy.

We previously reported altered neuronal Ca 2+ dynamics in the motor cortex of 12-month-old JNPL3 tauopathy mice during quiet wakefulness or forced running, with a tau antibody treatment significantly restoring the neuronal Ca 2+ activity profile and decreasing pathological tau in these mice 1 . Whether neuronal functional deficits occur at an early stage of tauopathy and if tau antibody treatment is effective in younger tauopathy mice needed further investigation. In addition, neuronal network activity and neuronal firing patterns have not been well studied in behaving tauopathy models. In this study, we first performed in vivo two-photon Ca 2+ imaging in JNPL3 mice in their early stage of tauopathy at 6 months of age, compared to 12 month old mice and age-matched wild-type controls to evaluate neuronal functional deficits. At the animal level, frequency of neuronal Ca 2+ transients decreased only in 6 month old tauopathy mice compared to controls, and only when animals were running on a treadmill. The amplitude of neuronal transients decreased in tauopathy mice compared to controls under resting and running conditions in both age groups. Total neuronal activity decreased only in 6 month old tauopathy mice compared to controls under resting and running conditions. Within either tauopathy or wild-type group, only total activity decreased in older wild-type animals. The tauopathy mice at different ages did not differ in neuronal Ca 2+ transient frequency, amplitude or total activity. In summary, neuronal function did significantly attenuate at an early age in tauopathy mice compared to controls but interestingly did not deteriorate between 6 and 12 months of age. A more detailed populational analysis of the pattern of Ca 2+ activity at the neuronal level in the 6 month old cohort confirmed neuronal hypoactivity in layer 2/3 of primary motor cortex, compared to wild-type controls, when animals were either resting or running on a treadmill. Despite reduced activity, neuronal Ca 2+ profiles exhibited enhanced synchrony and dysregulated responses to running stimulus. Further ex vivo electrophysiological recordings revealed reduction of spontaneous excitatory synaptic transmission onto and in pyramidal neurons and enhanced excitability of inhibitory neurons in motor cortex, which were likely responsible for altered neuronal network activity in this region. Lastly, tau antibody treatment reduced pathological tau and gliosis partially restored the neuronal Ca 2+ activity deficits but failed to rescue altered network changes. Taken together, substantial neuronal and network dysfunction occurred in the early stage of tauopathy that was partially alleviated with acute tau antibody treatment, which highlights the importance of functional assessment when evaluating the therapeutic potential of tau antibodies. Highlights: Layer 2/3 motor cortical neurons exhibited hypofunction in awake and behaving mice at the early stage of tauopathy.Altered neuronal network activity disrupted local circuitry engagement in tauopathy mice during treadmill running.Layer 2/3 motor cortical neurons in tauopathy mice exhibited enhanced neuronal excitability and altered excitatory synaptic transmissions.Acute tau antibody treatment reduced pathological tau and gliosis, and partially restored neuronal hypofunction profiles but not network dysfunction.

Tau-targeting therapies for Alzheimer disease: current status and future directions.

Alzheimer disease (AD) is the most common cause of dementia in older individuals. AD is characterized pathologically by amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the brain, with associated loss of synapses and neurons, which eventually results in dementia. Many of the early attempts to develop treatments for AD focused on Aß, but a lack of efficacy of these treatments in terms of slowing disease progression led to a change of strategy towards targeting of tau pathology. Given that tau shows a stronger correlation with symptom severity than does Aß, targeting of tau is more likely to be efficacious once cognitive decline begins. Anti-tau therapies initially focused on post-translational modifications, inhibition of tau aggregation and stabilization of microtubules. However, trials of many potential drugs were discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting agents in clinical trials are immunotherapies. In this Review, we provide an update on the results from the initial immunotherapy trials and an overview of new therapeutic candidates that are in clinical development, as well as considering future directions for tau-targeting therapies.

BAG3 Regulation of RAB35 Mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome Pathway and Tau Clearance.

BACKGROUND: Declining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimer's disease (AD). Emerging studies implicate impairment of the endosome-lysosome pathway as a significant factor in the pathogenesis of these diseases. Previously, we demonstrated that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression. METHODS: Mass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3-HSP70-TBC1D10B (EPI64B)-RAB35-HRS regulatory networks. Live-cell imaging was used to study the dynamics of the endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and in P301S tau transgenic mice with BAG3 overexpressed. RESULTS: The primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the RAB35 GTPase-activating protein TBC1D10B. We demonstrated that a BAG3-HSP70-TBC1D10B complex attenuates the ability of TBC1D10B to inactivate RAB35. Thus, BAG3 interacts with TBC1D10B to support the activation of RAB35 and recruitment of HRS, initiating endosomal sorting complex required for transport-mediated endosomal tau clearance. Furthermore, TBC1D10B shows significantly less colocalization with BAG3 in AD brains than in age-matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the colocalization of phosphorylated tau with the endosomal sorting complex required for transport III protein CHMP2B and reduced the levels of the mutant human tau. CONCLUSIONS: We identified a novel BAG3-TBC1D10B-RAB35 regulatory axis that modulates endosomal sorting complex required for transport-dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD.

Current Status of Clinical Trials on Tau Immunotherapies.

Tau immunotherapies have advanced from proof-of-concept studies to over a dozen clinical trials for Alzheimer's disease (AD) and other tauopathies. Mechanistic studies in animal and culture models have provided valuable insight into how these therapies may work but multiple pathways are likely involved. Different groups have emphasized the importance of intracellular vs extracellular antibody-mediated clearance of the tau protein and there is no consensus on which pool of tau should ideally be targeted. Likewise, various normal and disease-selective epitopes are being targeted, and the antibody isotypes either favor phagocytosis of the tau-antibody complex or are neutral in that aspect. Most of the clinical trials are in early stages, thus their efficacy is not yet known, but all have been without any major adverse effects and some have reported target engagement. A few have been discontinued. One in phase I, presumably because of a poor pharmacokinetic profile, and three in phase II for a lack of efficacy although this trial stage is not well powered for efficacy measures. In these phase II studies, trials with two antibodies in patients with progressive supranuclear palsy or other primary tauopathies were halted but are continuing in patients with AD, and one antibody trial was stopped in early-stage AD but is continuing in moderate AD. These three antibodies have been reported to only work extracellularly and tau is not increased in the cerebrospinal fluid of primary tauopathies, which may explain the failures of two of them. In the discontinued AD trial, there are some concerns about how much of extracellular tau contains the N-terminal epitope that is being targeted. In addition, extracellular tau is only a small part of total tau, compared to intracellular tau. Targeting only the former may not be sufficient for functional benefits. Given these outcomes, decision makers within the pharmaceutical companies who green light these trials should attempt to target tau not only extracellularly but also intracellularly to increase their chances of success. Hopefully, some of the ongoing trials will provide some functional benefits to the large number of patients with tauopathies.

Structural and functional characterization of the bestrophin-2 anion channel.

The bestrophin family of calcium (Ca2+)-activated chloride (Cl-) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca2+, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca2+ at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca2+, it has substantial Ca2+-independent activity for Cl-, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca2+ is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.

BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes.

A major cellular catabolic pathway in neurons is macroautophagy/autophagy, through which misfolded or aggregation-prone proteins are sequestered into autophagosomes that fuse with lysosomes, and are degraded. MAPT (microtubule-associated protein tau) is one of the protein clients of autophagy. Given that accumulation of hyperphosphorylated MAPT contributes to the pathogenesis of Alzheimer disease and other tauopathies, decreasing endogenous MAPT levels has been shown to be beneficial to neuronal health in models of these diseases. A previous study demonstrated that the HSPA/HSP70 co-chaperone BAG3 (BCL2-associated athanogene 3) facilitates endogenous MAPT clearance through autophagy. These findings prompted us to further investigate the mechanisms underlying BAG3-mediated autophagy in the degradation of endogenous MAPT. Here we demonstrate for the first time that BAG3 plays an important role in autophagic flux in the neurites of mature neurons (20-24 days in vitro [DIV]) through interaction with the post-synaptic cytoskeleton protein SYNPO (synaptopodin). Loss of either BAG3 or SYNPO impeded the fusion of autophagosomes and lysosomes predominantly in the post-synaptic compartment. A block of autophagy leads to accumulation of the autophagic receptor protein SQSTM1/p62 (sequestosome 1) as well as MAPT phosphorylated at Ser262 (p-Ser262). Furthermore, p-Ser262 appears to accumulate in autophagosomes at post-synaptic densities. Overall these data provide evidence of a novel role for the co-chaperone BAG3 in synapses. In cooperation with SYNPO, it functions as part of a surveillance complex that facilitates the autophagic clearance of MAPT p-Ser262, and possibly other MAPT species at the post-synapse. This appears to be crucial for the maintenance of a healthy, functional synapse.Abbreviations: aa: amino acids; ACTB: actin beta; BafA1: bafilomycin A1; BAG3: BCL2 associated athanogene 3; CQ chloroquine; CTSL: cathepsin L; DIV: days in vitro; DLG4/PSD95: discs large MAGUK scaffold protein 4; HSPA/HSP70: heat shock protein family A (Hsp70); MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP2: microtubule associated protein 2; MAPT: microtubule associated protein tau; p-Ser262: MAPT phosphorylated at serine 262; p-Ser396/404: MAPT phosphorylated at serines 396 and 404; p-Thr231: MAPT phosphorylated at threonine 231; PBS: phosphate buffered saline; PK: proteinase K; scr: scrambled; shRNA: short hairpin RNA; SQSTM1/p62 sequestosome 1; SYN1: synapsin I; SYNPO synaptopodin; SYNPO2/myopodin: synaptopodin 2; VPS: vacuolar protein sorting.

Dual Ca2+-dependent gates in human Bestrophin1 underlie disease-causing mechanisms of gain-of-function mutations.

Mutations of human BEST1, encoding a Ca2+-activated Cl- channel (hBest1), cause macular degenerative disorders. Best1 homolog structures reveal an evolutionarily conserved channel architecture highlighted by two landmark restrictions (named the "neck" and "aperture", respectively) in the ion conducting pathway, suggesting a unique dual-switch gating mechanism, which, however, has not been characterized well. Using patch clamp and crystallography, we demonstrate that both the neck and aperture in hBest1 are Ca2+-dependent gates essential for preventing channel leakage resulting from Ca2+-independent, spontaneous gate opening. Importantly, three patient-derived mutations (D203A, I205T and Y236C) lead to Ca2+-independent leakage and elevated Ca2+-dependent anion currents due to enhanced opening of the gates. Moreover, we identify a network of residues critically involved in gate operation. Together, our results suggest an indispensable role of the neck and aperture of hBest1 for channel gating, and uncover disease-causing mechanisms of hBest1 gain-of-function mutations.

Investigation and Restoration of BEST1 Activity in Patient-derived RPEs with Dominant Mutations.

BEST1 is a Ca2+-activated Cl- channel predominantly expressed in retinal pigment epithelium (RPE), and over 250 genetic mutations in the BEST1 gene have been identified to cause retinal degenerative disorders generally known as bestrophinopathies. As most BEST1 mutations are autosomal dominant, it is of great biomedical interest to determine their disease-causing mechanisms and the therapeutic potential of gene therapy. Here, we characterized six Best vitelliform macular dystrophy (BVMD)-associated BEST1 dominant mutations by documenting the patients' phenotypes, examining the subcellular localization of endogenous BEST1 and surface Ca2+-dependent Cl- currents in patient-derived RPEs, and analyzing the functional influences of these mutations on BEST1 in HEK293 cells. We found that all six mutations are loss-of-function with different levels and types of deficiencies, and further demonstrated the restoration of Ca2+-dependent Cl- currents in patient-derived RPE cells by WT BEST1 gene supplementation. Importantly, BEST1 dominant and recessive mutations are both rescuable at a similar efficacy by gene augmentation via adeno-associated virus (AAV), providing a proof-of-concept for curing the vast majority of bestrophinopathies.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

ATP activates bestrophin ion channels through direct interaction.

Human Bestrophin1 (hBest1) is a Ca2+-activated Cl- channel in retinal pigment epithelium (RPE) essential for retina physiology, and its mutation results in retinal degenerative diseases that have no available treatments. Here, we discover that hBest1's channel activity in human RPE is significantly enhanced by adenosine triphosphate (ATP) in a dose-dependent manner. We further demonstrate a direct interaction between ATP and bestrophins, and map the ATP-binding motif on hBest1 to an intracellular loop adjacent to the channel activation gate. Importantly, a disease-causing mutation of hBest1 located within the ATP-binding motif, p.I201T, diminishes ATP-dependent activation of the channel in patient-derived RPE, while the corresponding mutants in bestrophin homologs display defective ATP binding and a conformational change in the ATP-binding motif. Taken together, our results identify ATP as a critical activator of bestrophins, and reveal the molecular mechanism of an hBest1 patient-specific mutation.

Nrf2 mediates the expression of BAG3 and autophagy cargo adaptor proteins and tau clearance in an age-dependent manner.

During aging, decreased efficiency of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation and autophagic processes in the brain may be a contributing factor in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. Therefore, we analyzed the expression of Bcl-2-associated athanogene 3, a cochaperone that mediates autophagy, and the autophagy adaptors NBR1, NDP52, and sequestosome 1/p62 in the brains of 4-, 8-, and 12-month-old wild-type and Nrf2 knockout (-/-) mice. We also analyzed the levels of total tau and phospho-tau species. There were minimal differences in the expression of autophagy-related genes or tau species in 4-month-old animals; however, by 12 months, all of these autophagy-associated genes were expressed at significantly lower levels in the Nrf2 (-/-) mice. The decreases in the autophagy-associated genes were accompanied by significantly elevated levels of phospho-tau species in the 12-month-old Nrf2 (-/-) brains. These findings indicate that Nrf2 regulation of autophagy-related genes likely plays a greater role in mediating the clearance of tau as an organism ages.

Assessing the degradation of tau in primary neurons: The role of autophagy.

Tau is a neuronal cytosolic, highly regulated protein. Although first identified as a protein that binds and stabilizes microtubules, it is now clear that tau plays numerous other roles in neurons. In addition to its key physiological roles in neuronal structure and function, tau is also involved in the pathogenesis of Alzheimer's disease and numerous other neurodegenerative disorders. In all tauopathies, there are pathogenic accumulations of tau. Given that tau homeostasis requires a balance of synthesis and degradation, understanding the pathways that mediate tau clearance and regulate this process in the disease state is of fundamental importance. In neurons, macroautophagy (referred to as autophagy in this chapter) plays a pivotal role in clearing damaged or misfolded proteins under normal conditions. However, in the disease state autophagy is impaired and tau may not be efficiently targeted for degradation which contributes to the increases in pathological tau species. Therefore, establishing model systems that allow for the analysis of tau clearance by autophagy and quantitative assessment of interventions that increase autophagy and tau clearance are needed. Of particular importance is the use of primary neurons as a model system, as they are more reflective of the relevant in vivo autophagy pathway than clonal or immortalized cell models. In this chapter we present detailed methods for the preparation of neurons, immunoblotting and imaging analyses, genetic and pharmacological manipulation of autophagy with analyses, and methods to quantitatively measure changes in tau and phospho-tau levels.