Caspase-3 activation via mitochondria is required for long-term depression and AMPA receptor internalization.

NMDA receptor-dependent synaptic modifications, such as long-term potentiation (LTP) and long-term depression (LTD), are essential for brain development and function. LTD occurs mainly by the removal of AMPA receptors from the postsynaptic membrane, but the underlying molecular mechanisms remain unclear. Here, we show that activation of caspase-3 via mitochondria is required for LTD and AMPA receptor internalization in hippocampal neurons. LTD and AMPA receptor internalization are blocked by peptide inhibitors of caspase-3 and -9. In hippocampal slices from caspase-3 knockout mice, LTD is abolished whereas LTP remains normal. LTD is also prevented by overexpression of the anti-apoptotic proteins XIAP or Bcl-xL, and by a mutant Akt1 protein that is resistant to caspase-3 proteolysis. NMDA receptor stimulation that induces LTD transiently activates caspase-3 in dendrites, without causing cell death. These data indicate an unexpected causal link between the molecular mechanisms of apoptosis and LTD.

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

BACKGROUND: Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. METHODS: We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. RESULTS: Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6-/- podocytes. CONCLUSIONS: Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.

Glucocorticoids activate a synapse weakening pathway culminating in tau phosphorylation in the hippocampus.

Evidence suggests that the stress hormones glucocorticoids (GCs) can cause cognitive deficits and neurodegeneration. Previous studies have found GCs facilitate physiological synapse weakening, termed long-term depression (LTD), though the precise mechanisms underlying this are poorly understood. Here we show that GCs activate glycogen synthase kinase-3 (GSK-3), a kinase crucial to synapse weakening signals. Critically, this ultimately leads to phosphorylation of the microtubule associated protein tau, specifically at the serine 396 residue, and this is a causal factor in the GC-mediated impairment of synaptic function. These findings reveal the link between GCs and synapse weakening signals, and the potential for stress-induced priming of neurodegeneration. This could have important implications for our understanding of how stress can lead to neurodegenerative disease.

Tau phosphorylation at serine 396 residue is required for hippocampal LTD.

Tau is required for the induction of long-term depression (LTD) of synaptic transmission in the hippocampus. Here we probe the role of tau in LTD, finding that an AMPA receptor internalization mechanism is impaired in tau KO mice, and that LTD causes specific phosphorylation at the serine 396 and 404 residues of tau. Surprisingly, we find that phosphorylation at serine 396, specifically, is critical for LTD but has no role in LTP. Finally, we show that tau KO mice exhibit deficits in spatial reversal learning. These findings underscore the physiological role for tau at the synapse and identify a behavioral correlate of its role in LTD.

Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF).

This study describes a fundamental functional difference between the two main polymorphisms of the pro-form of brain-derived neurotrophic factor (proBDNF), providing an explanation as to why these forms have such different age-related neurological outcomes. Healthy young carriers of the Met66 form (present in ∼30% Caucasians) have reduced hippocampal volume and impaired hippocampal-dependent memory function, yet the same polymorphic population shows enhanced cognitive recovery after traumatic brain injury, delayed cognitive dysfunction during aging, and lower risk of late-onset Alzheimer's disease (AD) compared to those with the more common Val66 polymorphism. To examine the differences between the protein polymorphisms in structure, kinetics of binding to proBDNF receptors and in vitro function, we generated purified cleavage-resistant human variants. Intriguingly, we found no statistical differences in those characteristics. As anticipated, exogenous application of proBDNF Val66 to rat hippocampal slices dysregulated synaptic plasticity, inhibiting long-term potentiation (LTP) and facilitating long-term depression (LTD). We subsequently observed that this occurred via the glycogen synthase kinase 3ß (GSK3ß) activation pathway. However, surprisingly, we found that Met66 had no such effects on either LTP or LTD. These novel findings suggest that, unlike Val66, the Met66 variant does not facilitate synapse weakening signaling, perhaps accounting for its protective effects with aging.

Is riluzole a new drug for Alzheimer's disease?

This Editorial highlights a study by Hunsberger et al. (2015) in the current issue of Journal of Neurochemistry, in which the authors explore the effects of riluzole (R) treatment on tau-P301L transgenic mice. The authors employed a comprehensive analysis of possible restorative effects of the drug by examining glutamate levels in subregions of the hippocampus, expression of tau and its hyper-phosphorylated forms, and memory function using behavioral tests. The authors report a simultaneous reduction in glutamate reuptake and an increase in glutamate release in the tau-P301L model, both of which are ameliorated with riluzole treatment. The authors' findings have implications for our understanding of synaptic transmission mechanisms also associated with Alzheimer's disease pathology.

Rare individual amyloid-ß oligomers act on astrocytes to initiate neuronal damage.

Oligomers of the amyloid-ß (Aß) peptide have been implicated in the neurotoxicity associated with Alzheimer's disease. We have used single-molecule techniques to examine quantitatively the cellular effects of adding well characterized Aß oligomers to primary hippocampal cells and hence determine the initial pathway of damage. We found that even picomolar concentrations of Aß (1-40) and Aß (1-42) oligomers can, within minutes of addition, increase the levels of intracellular calcium in astrocytes but not in neurons, and this effect is saturated at a concentration of about 10 nM of oligomers. Both Aß (1-40) and Aß (1-42) oligomers have comparable effects. The rise in intracellular calcium is followed by an increase in the rate of ROS production by NADPH oxidase in both neurons and astrocytes. The increase in ROS production then triggers caspase-3 activation resulting in the inhibition of long-term potentiation. Our quantitative approach also reveals that only a small fraction of the oligomers are damaging and that an individual rare oligomer binding to an astrocyte can initiate the aforementioned cascade of responses, making it unlikely to be due to any specific interaction. Preincubating the Aß oligomers with an extracellular chaperone, clusterin, sequesters the oligomers in long-lived complexes and inhibits all of the physiological damage, even at a ratio of 100:1, total Aß to clusterin. To explain how Aß oligomers are so damaging but that it takes decades to develop Alzheimer's disease, we suggest a model for disease progression where small amounts of neuronal damage from individual unsequestered oligomers can accumulate over time leading to widespread tissue-level dysfunction.

Acute stress causes rapid synaptic insertion of Ca2+ -permeable AMPA receptors to facilitate long-term potentiation in the hippocampus.

The neuroendocrine response to episodes of acute stress is crucial for survival whereas the prolonged response to chronic stress can be detrimental. Learning and memory are particularly susceptible to stress with cognitive deficits being well characterized consequences of chronic stress. Although there is good evidence that acute stress can enhance cognitive performance, the mechanism(s) for this are unclear. We find that hippocampal slices, either prepared from rats following 30 min restraint stress or directly exposed to glucocorticoids, exhibit an N-methyl-d-aspartic acid receptor-independent form of long-term potentiation. We demonstrate that the mechanism involves an NMDA receptor and PKA-dependent insertion of Ca2+ -permeable AMPA receptors into synapses. These then trigger the additional NMDA receptor-independent form of LTP during high frequency stimulation.

Sensing change: the emerging role of calcium sensors in neuronal disease.

Calcium (Ca(2+)) is a fundamental intracellular signalling molecule in neurons. Therefore, significant interest has been expressed in understanding how the dysregulation of Ca(2+) signals might impact on neuronal function and the progression of different disease states. Many previous studies have examined the role of Ca(2+) in neuronal excitotoxicity and some have started to understand how Ca(2+) dysregulation might be a cause or consequence of neurodegeneration. This review will therefore focus on the significance of Ca(2+) sensors, proteins that transduce Ca(2+) signals, in neuronal function and dysfunction. Finally, we will assess their potential role in neurodegenerative processes, such as Alzheimer's disease (AD), arguing that they could serve as potential therapeutic targets.

False recognition in a mouse model of Alzheimer's disease: rescue with sensory restriction and memantine.

Alzheimer's disease is commonly regarded as a loss of memory for past events. However, patients with Alzheimer's disease seem not only to forget events but also to express false confidence in remembering events that have never happened. How and why false recognition occurs in such patients is currently unknown, and treatments targeting this specific mnemonic abnormality have not been attempted. Here, we used a modified object recognition paradigm to show that the tgCRND8 mouse-which overexpresses amyloid ß and develops amyloid plaques similar to those in the brains of patients with Alzheimer's disease-exhibits false recognition. Furthermore, we found that false recognition did not occur when tgCRND8 mice were kept in a dark, quiet chamber during the delay, paralleling previous findings in patients with mild cognitive impairment, which is often considered to be prodromal Alzheimer's disease. Additionally, false recognition did not occur when mice were treated with the partial N-methyl-d-aspartic acid receptor antagonist memantine. In a subsequent experiment, we found abnormally enhanced N-methyl-d-aspartic acid receptor-dependent long-term depression in these mice, which could be normalized by treatment with memantine. We suggest that Alzheimer's disease typical amyloid ß pathology leads to aberrant synaptic plasticity, thereby making memory representations more susceptible to interfering sensory input, thus increasing the likelihood of false recognition. Parallels between these findings and those from the literature on Alzheimer's disease and mild cognitive impairment suggest a mechanism underlying false recognition in these patients. The false recognition phenomenon may provide a novel paradigm for the discovery of potential therapies to treat the mnemonic dysfunction characteristic of this disease.

Human stem cell-derived ventral midbrain astrocytes exhibit a region-specific secretory profile.

This scientific commentary refers to 'Human stem cell-derived astrocytes exhibit region-specific heterogeneity in their secretory profiles', by Clarke et al. (https://doi.org/10.1093/brain/awaa258) in Brain.

Mimicking hypomethylation of FUS requires liquid-liquid phase separation to induce synaptic dysfunctions.

The hypomethylation of fused in sarcoma (FUS) in frontotemporal lobar degeneration promotes the formation of irreversible condensates of FUS. However, the mechanisms by which these hypomethylated FUS condensates cause neuronal dysfunction are unknown. Here we report that expression of FUS constructs mimicking hypomethylated FUS causes aberrant dendritic FUS condensates in CA1 neurons. These hypomethylated FUS condensates exhibit spontaneous, and activity induced movement within the dendrite. They impair excitatory synaptic transmission, postsynaptic density-95 expression, and dendritic spine plasticity. These neurophysiological defects are dependent upon both the dendritic localisation of the condensates, and their ability to undergo liquid-liquid phase separation. These results indicate that the irreversible liquid-liquid phase separation is a key component of hypomethylated FUS pathophysiology in sporadic FTLD, and this can cause synapse dysfunction in sporadic FTLD.

Ultrasound modulates neuronal potassium currents via ionotropic glutamate receptors.

BACKGROUND: Focused ultrasound stimulation (FUS) has the potential to provide non-invasive neuromodulation of deep brain regions with unparalleled spatial precision. However, the cellular and molecular consequences of ultrasound stimulation on neurons remains poorly understood. We previously reported that ultrasound stimulation induces increases in neuronal excitability that persist for hours following stimulation in vitro. In the present study we sought to further elucidate the molecular mechanisms by which ultrasound regulates neuronal excitability and synaptic function. OBJECTIVES: To determine the effect of ultrasound stimulation on voltage-gated ion channel function and synaptic plasticity. METHODS: Primary rat cortical neurons were exposed to a 40 s, 200 kHz pulsed ultrasound stimulus or sham-stimulus. Whole-cell patch clamp electrophysiology, quantitative proteomics and high-resolution confocal microscopy were employed to determine the effects of ultrasound stimulation on molecular regulators of neuronal excitability and synaptic function. RESULTS: We find that ultrasound exposure elicits sustained but reversible increases in whole-cell potassium currents. In addition, we find that ultrasound exposure activates synaptic signalling cascades that result in marked increases in excitatory synaptic transmission. Finally, we demonstrate the requirement of ionotropic glutamate receptor (AMPAR/NMDAR) activation for ultrasound-induced modulation of neuronal potassium currents. CONCLUSION: These results suggest specific patterns of pulsed ultrasound can induce contemporaneous enhancement of both neuronal excitability and synaptic function, with implications for the application of FUS in experimental and therapeutic settings. Further study is now required to deduce the precise molecular mechanisms through which these changes occur.

AZD5438 a GSK-3a/b and CDK inhibitor is antiapoptotic modulates mitochondrial activity and protects human neurons from mitochondrial toxins.

We previously reported that kenpaullone, which inhibits GSK-3a/b and CDKs inhibited CCCP mediated mitochondrial depolarisation and augments the mitochondrial network. To investigate the actions of this class of drug further, we compared the ability of kenpaullone, alsterpaullone, 1-azakenapaullone, AZD5438, AT7519 (CDK and GSK-3a/b inhibitors) and dexpramipexole and olesoxime (mitochondrial permeability transition pore inhibitors) to prevent CCCP mediated mitochondrial depolarisation and found that AZD5438 and AT7519, were the most effective. Furthermore, treatment with AZD5438 alone increased the complexity of the mitochondrial network. We also found that AZD5438 prevented the rotenone induced decrease in PGC-1alpha and TOM20 levels and that it mediated powerful anti-apoptotic effects and promoted glycolytic respiration. Importantly, experiments in human iPSC derived cortical and midbrain neurons showed AZD5438 mediated significant protective effects, preventing the neuronal cell death, and collapse in the neurite and mitochondrial network associated with rotenone treatment. These results suggest drugs that target GSK-3a/b and CDKs should be developed and assessed further as they may have significant therapeutic potential.

Focused Ultrasound Stimulation as a Neuromodulatory Tool for Parkinson's Disease: A Scoping Review.

Non-invasive focused ultrasound stimulation (FUS) is a non-ionising neuromodulatory technique that employs acoustic energy to acutely and reversibly modulate brain activity of deep-brain structures. It is currently being investigated as a potential novel treatment for Parkinson's disease (PD). This scoping review was carried out to map available evidence pertaining to the provision of FUS as a PD neuromodulatory tool. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews, a search was applied to Ovid MEDLINE, Embase, Web of Science and Cochrane Central Register of Controlled Trials on 13 January 2022, with no limits applied. In total, 11 studies were included: 8 were from China and 1 each from Belgium, South Korea and Taiwan. All 11 studies were preclinical (6 in vivo, 2 in vitro, 2 mix of in vivo and in vitro and 1 in silico). The preclinical evidence indicates that FUS is safe and has beneficial neuromodulatory effects on motor behaviour in PD. FUS appears to have a therapeutic role in influencing the disease processes of PD, and therefore holds great promise as an attractive and powerful neuromodulatory tool for PD. Though these initial studies are encouraging, further study to understand the underlying cellular and molecular mechanisms is required before FUS can be routinely used in PD.

Transcriptional programs regulating neuronal differentiation are disrupted in DLG2 knockout human embryonic stem cells and enriched for schizophrenia and related disorders risk variants.

Coordinated programs of gene expression drive brain development. It is unclear which transcriptional programs, in which cell-types, are affected in neuropsychiatric disorders such as schizophrenia. Here we integrate human genetics with transcriptomic data from differentiation of human embryonic stem cells into cortical excitatory neurons. We identify transcriptional programs expressed during early neurogenesis in vitro and in human foetal cortex that are down-regulated in DLG2-/- lines. Down-regulation impacted neuronal differentiation and maturation, impairing migration, morphology and action potential generation. Genetic variation in these programs is associated with neuropsychiatric disorders and cognitive function, with associated variants predominantly concentrated in loss-of-function intolerant genes. Neurogenic programs also overlap schizophrenia GWAS enrichment previously identified in mature excitatory neurons, suggesting that pathways active during prenatal cortical development may also be associated with mature neuronal dysfunction. Our data from human embryonic stem cells, when combined with analysis of available foetal cortical gene expression data, de novo rare variants and GWAS statistics for neuropsychiatric disorders and cognition, reveal a convergence on transcriptional programs regulating excitatory cortical neurogenesis.

Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins.

Rho family small GTPases are important regulators of neuronal development. Defective Rho regulation causes nervous system dysfunctions including mental retardation and Alzheimer's disease. Rac1, a member of the Rho family, regulates dendritic spines and excitatory synapses, but relatively little is known about how synaptic Rac1 is negatively regulated. Breakpoint cluster region (BCR) is a Rac GTPase-activating protein known to form a fusion protein with the c-Abl tyrosine kinase in Philadelphia chromosome-positive chronic myelogenous leukemia. Despite the fact that BCR mRNAs are abundantly expressed in the brain, the neural functions of BCR protein have remained obscure. We report here that BCR and its close relative active BCR-related (ABR) localize at excitatory synapses and directly interact with PSD-95, an abundant postsynaptic scaffolding protein. Mice deficient for BCR or ABR show enhanced basal Rac1 activity but only a small increase in spine density. Importantly, mice lacking BCR or ABR exhibit a marked decrease in the maintenance, but not induction, of long-term potentiation, and show impaired spatial and object recognition memory. These results suggest that BCR and ABR have novel roles in the regulation of synaptic Rac1 signaling, synaptic plasticity, and learning and memory, and that excessive Rac1 activity negatively affects synaptic and cognitive functions.

Transient ultrasound stimulation has lasting effects on neuronal excitability.

BACKGROUND: Transcranial ultrasound stimulation can acutely modulate brain activity, but the lasting effects on neurons are unknown. OBJECTIVE: To assess the excitability profile of neurons in the hours following transient ultrasound stimulation. METHODS: Primary rat cortical neurons were stimulated with a 40 s, 200 kHz pulsed ultrasound stimulation or sham-stimulation. Intrinsic firing properties were investigated through whole-cell patch-clamp recording by evoking action potentials in response to somatic current injection. Recordings were taken at set timepoints following ultrasound stimulation: 0-2 h, 6-8 h, 12-14 h and 24-26 h. Transmission electron microscopy was used to assess synaptic ultrastructure at the same timepoints. RESULTS: In the 0-2 h window, neurons stimulated with ultrasound displayed an increase in the mean frequency of evoked action potentials of 32% above control cell levels (p = 0.023). After 4-6 h this increase was measured as 44% (p = 0.0043). By 12-14 h this effect was eliminated and remained absent 24-26 h post-stimulation. These changes to action potential firing occurred in conjunction with statistically significant differences between control and ultrasound-stimulated neurons in action potential half-width, depolarisation rate, and repolarisation rate, that were similarly eliminated by 24 h following stimulation. These effects occurred in the absence of alterations to intrinsic membrane properties or synaptic ultrastructure. CONCLUSION: We report that stimulating neurons with 40 s of ultrasound enhances their excitability for up to 8 h in conjunction with modifications to action potential kinetics. This occurs in the absence of major ultrastructural change or modification of intrinsic membrane properties. These results can inform the application of transcranial ultrasound in experimental and therapeutic settings.

M1 muscarinic acetylcholine receptor dysfunction in moderate Alzheimer's disease pathology.

Aggregation of amyloid beta and loss of cholinergic innervation in the brain are predominant components of Alzheimer's disease pathology and likely underlie cognitive impairment. Acetylcholinesterase inhibitors are one of the few treatment options for Alzheimer's disease, where levels of available acetylcholine are enhanced to counteract the cholinergic loss. However, these inhibitors show limited clinical efficacy. One potential explanation for this is a concomitant dysregulation of cholinergic receptors themselves as a consequence of the amyloid beta pathology. We tested this hypothesis by examining levels of M1 muscarinic acetylcholine receptors in the temporal cortex from seven Alzheimer's disease and seven non-disease age-matched control brain tissue samples (control: 85 ± 2.63 years old, moderate Alzheimer's disease: 84 ± 2.32 years old, P-value = 0.721; eight female and six male patients). The samples were categorized into two groups: 'control' (Consortium to Establish a Registry for Alzheimer's Disease diagnosis of 'No Alzheimer's disease', and Braak staging pathology of I-II) and 'moderate Alzheimer's disease' (Consortium to Establish a Registry for Alzheimer's Disease diagnosis of 'possible/probable Alzheimer's disease', and Braak staging pathology of IV). We find that in comparison to age-matched controls, there is a loss of M1 muscarinic acetylcholine receptors in moderate Alzheimer's disease tissue (control: 2.17 ± 0.27 arbitrary units, n = 7, Mod-AD: 0.83 ± 0.16 arbitrary units, n = 7, two-tailed t-test, t = 4.248, P = 0.00113). Using a functional rat cortical brain slice model, we find that postsynaptic muscarinic acetylcholine receptor function is dysregulated by aberrant amyloid beta-mediated activation of metabotropic glutamate receptor 5. Crucially, blocking metabotropic glutamate receptor 5 restores muscarinic acetylcholine receptor function and object recognition memory in 5XFAD transgenic mice. This indicates that the amyloid beta-mediated activation of metabotropic glutamate receptor 5 negatively regulates muscarinic acetylcholine receptor and illustrates the importance of muscarinic acetylcholine receptors as a potential disease-modifying target in the moderate pathological stages of Alzheimer's disease.