Effects of amyloid-beta on homeostatic network plasticity in human iPSC-derived neuronal networks

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder and it is the most common cause of dementia in elderly. The disease is characterized by memory loss, mood swings, and communication problems. This uniquely human disease has been investigated in various mouse models mimicking different pathological hallmarks of AD, which supplied valuable insight into disease mechanisms; however, clinical trials based on these models failed and current treatments are unsatisfactory. To overcome the limitations of animal models of AD, the emerging induced pluripotent stem cell (iPSC) technology promises great potential. It offers the possibility to investigate underlying disease mechanisms, screen for drug targets and validate therapeutic effects in disease-relevant cell types of human origin on a patient-specific background. Current iPSC studies to model AD have been addressing the questions of the pathological hallmarks such as an increase in amyloid beta (Aß) and hyperphosphorylated tau. However, to investigate the AD-related phenomenon of neuronal hyperactivity, mature human neuronal cultures with spontaneously active networks are necessary, and their generation remains a challenge. In this study, to achieve spontaneously firing mature neuronal networks, human iPS cells were differentiated into neurons and were supported with endogenously differentiated human astrocytes or primary cortical astrocytes (PCA) isolated from rat brains. Neuronal activity was recorded by using multi electrode array (MEA) to detect single spikes and network bursts. Calcium imaging of spontaneously firing networks was performed to monitor synchronously active neurons in cultures. To trigger hyperactivity-induced homeostatic plasticity in human networks, iPSC-derived cultures were treated with 4-Aminopyridine (4AP), a non-selective inhibitor of voltage-gated K+ channels. It increased the network activity only in mature (burst firing) cultures. This induced hyperactivity further led to activation of homeostatic plasticity dependent mechanisms to reduce the firing rate. Single spike analysis suggested Na+ channel removal from the axonal membrane as one of these compensatory mechanisms. Moreover, repressor element-1 silencing transcription factor (REST) was identified as a key player in this process. To study AD-related impairments in the established model, cell-derived and synthetic Aß oligomers were prepared and characterized by semi-native western blot. Synthetic Aß oligomers were surprisingly stable when added to neuronal cultures and caused no cell death and no change in spontaneous network activity. However, upon 4AP treatment, Aß-treated networks showed impaired homeostatic plasticity and were not able to reduce the firing rate appropriately. According to the analysis of spike properties, the plasticity-associated reduction of axonal Na+ channels was also impaired. In Aß-treated cultures, nuclear REST expression was diminished at basal levels and after triggering homeostatic plasticity by 4AP. Thus, AD-related hyperactivity may be caused by dysfunctional homeostatic plasticity in a REST-dependent manner. Taken together, the results of this study provided the first hint on a previously unknown impairment of homeostatic plasticity mechanisms in AD and identified REST as a target which might contribute to the hyperactivity phenomenon at early stages of AD. This knowledge of plasticity impairment might expand our understanding of disease development and REST manipulation might be a new target for potential therapeutic strategies

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies

Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed. RESULTS: To study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2-3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture's genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions. CONCLUSION: The successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments

Effects of amyloid-beta on homeostatic network plasticity in human iPSC-derived neuronal networks

Alzheimer’s disease (AD) is a progressive, neurodegenerative disorder and it is the most common cause of dementia in elderly. The disease is characterized by memory loss, mood swings, and communication problems. This uniquely human disease has been investigated in various mouse models mimicking different pathological hallmarks of AD, which supplied valuable insight into disease mechanisms; however, clinical trials based on these models failed and current treatments are unsatisfactory. To overcome the limitations of animal models of AD, the emerging induced pluripotent stem cell (iPSC) technology promises great potential. It offers the possibility to investigate underlying disease mechanisms, screen for drug targets and validate therapeutic effects in disease-relevant cell types of human origin on a patient-specific background. Current iPSC studies to model AD have been addressing the questions of the pathological hallmarks such as an increase in amyloid beta (Aß) and hyperphosphorylated tau. However, to investigate the AD-related phenomenon of neuronal hyperactivity, mature human neuronal cultures with spontaneously active networks are necessary, and their generation remains a challenge. In this study, to achieve spontaneously firing mature neuronal networks, human iPS cells were differentiated into neurons and were supported with endogenously differentiated human astrocytes or primary cortical astrocytes (PCA) isolated from rat brains. Neuronal activity was recorded by using multi electrode array (MEA) to detect single spikes and network bursts. Calcium imaging of spontaneously firing networks was performed to monitor synchronously active neurons in cultures. To trigger hyperactivity-induced homeostatic plasticity in human networks, iPSC-derived cultures were treated with 4-Aminopyridine (4AP), a non-selective inhibitor of voltage-gated K+ channels. It increased the network activity only in mature (burst firing) cultures. This induced hyperactivity further led to activation of homeostatic plasticity dependent mechanisms to reduce the firing rate. Single spike analysis suggested Na+ channel removal from the axonal membrane as one of these compensatory mechanisms. Moreover, repressor element-1 silencing transcription factor (REST) was identified as a key player in this process. To study AD-related impairments in the established model, cell-derived and synthetic Aß oligomers were prepared and characterized by semi-native western blot. Synthetic Aß oligomers were surprisingly stable when added to neuronal cultures and caused no cell death and no change in spontaneous network activity. However, upon 4AP treatment, Aß-treated networks showed impaired homeostatic plasticity and were not able to reduce the firing rate appropriately. According to the analysis of spike properties, the plasticity-associated reduction of axonal Na+ channels was also impaired. In Aß-treated cultures, nuclear REST expression was diminished at basal levels and after triggering homeostatic plasticity by 4AP. Thus, AD-related hyperactivity may be caused by dysfunctional homeostatic plasticity in a REST-dependent manner. Taken together, the results of this study provided the first hint on a previously unknown impairment of homeostatic plasticity mechanisms in AD and identified REST as a target which might contribute to the hyperactivity phenomenon at early stages of AD. This knowledge of plasticity impairment might expand our understanding of disease development and REST manipulation might be a new target for potential therapeutic strategies

Prominent microglial inclusions in transgenic mouse models of α-synucleinopathy that are distinct from neuronal lesions.

Alpha-synucleinopathies are a group of progressive neurodegenerative disorders, characterized by intracellular deposits of aggregated α-synuclein (αS). The clinical heterogeneity of these diseases is thought to be attributed to conformers (or strains) of αS but the contribution of inclusions in various cell types is unclear. The aim of the present work was to study αS conformers among different transgenic (TG) mouse models of α-synucleinopathies. To this end, four different TG mouse models were studied (Prnp-h[A53T]αS; Thy1-h[A53T]αS; Thy1-h[A30P]αS; Thy1-mαS) that overexpress human or murine αS and differed in their age-of-symptom onset and subsequent disease progression. Postmortem analysis of end-stage brains revealed robust neuronal αS pathology as evidenced by accumulation of αS serine 129 (p-αS) phosphorylation in the brainstem of all four TG mouse lines. Overall appearance of the pathology was similar and only modest differences were observed among additionally affected brain regions. To study αS conformers in these mice, we used pentameric formyl thiophene acetic acid (pFTAA), a fluorescent dye with amyloid conformation-dependent spectral properties. Unexpectedly, besides the neuronal αS pathology, we also found abundant pFTAA-positive inclusions in microglia of all four TG mouse lines. These microglial inclusions were also positive for Thioflavin S and showed immunoreactivity with antibodies recognizing the N-terminus of αS, but were largely p-αS-negative. In all four lines, spectral pFTAA analysis revealed conformational differences between microglia and neuronal inclusions but not among the different mouse models. Concomitant with neuronal lesions, microglial inclusions were already present at presymptomatic stages and could also be induced by seeded αS aggregation. Although nature and significance of microglial inclusions for human α-synucleinopathies remain to be clarified, the previously overlooked abundance of microglial inclusions in TG mouse models of α-synucleinopathy bears importance for mechanistic and preclinical-translational studies