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The endoplasmic reticulum (ER)’s continuous morphology is tightly controlled by ER‐shaping proteins, whose genetic or expression defects drive a spectrum of neurodegenerative disorders from Hereditary Spastic Paraplegia to Alzheimer's disease. Why perturbations in ER morphology manifest specifically in neurons remains unknown. Here, by coupling visualisation of global sub‐Hz firing bursts to ER ultrastructural manipulations in human inducible Pluripotent Stem Cells (hiPSC)‐derived cortical neurons, alongside physical simulations, we establish a key ER structure‐function principle: neuronal ER architecture dictates Ca2+ replenishment speed. Altering ER structure hinders network ER luminal connectivity and Ca2+ propagation from refill points at plasma membrane contact sites, impairing the ER's capability to supply repetitive Ca2+ bursts. The ER morpho‐regulatory control of Ca2+ refill speed thus constitutes a switch on neuronal activity. Further, perturbed ER shape also abolishes Ca2+ firing and contraction in primary skeletal muscle cells. These results expose the selective vulnerability of Ca2+‐firing cells to ER structural disruptions, rationalizing ER dysfunction in neurodegeneration and unveiling a new role for the continuous ER morphology that could apply universally to Ca2+‐firing cells. The ER's continuous tubular network is maintained by ER‐shaping proteins whose mutation or dysregulation contributes to neurodegenerative diseases. Here, we show that ER morphology sets the speed of Ca2+ store replenishment between firing events. Disrupting ER continuity slows intra‐ER Ca2+ redistribution from extracellular refill (SOCE) sites, driving burst rundown and functional failure—offering a mechanism for selective neuronal vulnerability to ER defects.

Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer’s disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

Cognitive impairment is one of the many symptoms reported by individuals suffering from long-COVID and other post-viral infection disorders such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). A common factor among these conditions is a sustained immune response and increased levels of inflammatory cytokines. Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are two such cytokines that are elevated in patients diagnosed with long-COVID and ME/CFS. In this study, we characterized the changes in neural functionality, secreted cytokine profiles, and gene expression in co-cultures of human iPSC-derived neurons and primary astrocytes in response to prolonged exposure to TNF-α and IL-6. We found that exposure to TNF-α produced both a concentration-independent and concentration-dependent response in neural activity. Burst duration was significantly reduced within a few days of exposure regardless of concentration (1 pg/mL – 100 ng/mL) but returned to baseline after 7 days. Treatment with low concentrations of TNF-α (e.g., 1 and 25 pg/mL) did not lead to changes in the secreted cytokine profile or gene expression but still resulted in significant changes to electrophysiological features such as interspike interval and burst duration. Conversely, treatment with high concentrations of TNF-α (e.g., 10 and 100 ng/mL) led to reduced spiking activity, which may be correlated to changes in neural health, gene expression, and increases in inflammatory cytokine secretion (e.g., IL-1β, IL-4, and CXCL-10) that were observed at higher TNF-α concentrations. Prolonged exposure to IL-6 led to changes in bursting features, with significant reduction in the number of spikes in bursts across a wide range of treatment concentrations (i.e., 1 pg/mL–10 ng/mL). In combination, the addition of IL-6 appears to counteract the changes to neural function induced by low concentrations of TNF-α, while at high concentrations of TNF-α the addition of IL-6 had little to no effect. Conversely, the changes to electrophysiological features induced by IL-6 were lost when the cultures were co-stimulated with TNF-α regardless of the concentration, suggesting that TNF-α may play a more pronounced role in altering neural function. These results indicate that increased concentrations of key inflammatory cytokines associated with long-COVID can directly impact neural function and may be a component of the cognitive impairment associated with long-COVID and other post-viral infection disorders.

Batten disease is a devastating, childhood, rare neurodegenerative disease characterised by the rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene, leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed reduced acidic organelles and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement—a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections, and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

Besides respiratory illness, SARS-CoV-2, the causative agent of COVID-19, leads to neurological symptoms. The molecular mechanisms leading to neuropathology after SARS-CoV-2 infection are sparsely explored. SARS-CoV-2 enters human cells via different receptors, including ACE-2, TMPRSS2, and TMEM106B. In this study, we used a human-induced pluripotent stem cell-derived neuronal model, which expresses ACE-2, TMPRSS2, TMEM106B, and other possible SARS-CoV-2 receptors, to evaluate its susceptibility to SARS-CoV-2 infection. The neurons were exposed to SARS-CoV-2, followed by RT-qPCR, immunocytochemistry, and proteomic analyses of the infected neurons. Our findings showed that SARS-CoV-2 infects neurons at a lower rate than other human cells; however, the virus could not replicate or produce infectious virions in this neuronal model. Despite the aborted SARS-CoV-2 replication, the infected neuronal nuclei showed irregular morphology compared to other human cells. Since cytokine storm is a significant effect of SARS-CoV-2 infection in COVID-19 patients, in addition to the direct neuronal infection, the neurons were treated with pre-conditioned media from SARS-CoV-2-infected lung cells, and the neuroproteomic changes were investigated. The limited SARS-CoV-2 infection in the neurons and the neurons treated with the pre-conditioned media showed changes in the neuroproteomic profile, particularly affecting mitochondrial proteins and apoptotic and metabolic pathways, which may lead to the development of neurological complications. The findings from our study uncover a possible mechanism behind SARS-CoV-2-mediated neuropathology that might contribute to the lingering effects of the virus on the human brain.

Synapse loss is the principal cause of cognitive decline in Alzheimer’s disease (AD) and related disorders (ADRD). Synapse development depends on the intricate dynamics of the neuronal cytoskeleton. Cofilin, the major protein regulating actin dynamics, can be sequestered into cofilactin rods, intra-neurite bundles of cofilin-saturated actin filaments that can disrupt vesicular trafficking and cause synaptic loss. Rods are a brain pathology in human AD and mouse models of AD and ADRD. Eliminating rods is the focus of this paper. One pathway for rod formation is triggered in ~20% of rodent hippocampal neurons by disease-related factors (e.g., soluble oligomers of Amyloid-β (Aβ)) and requires cellular prion protein (PrPC), active NADPH oxidase (NOX), and cytokine/chemokine receptors (CCRs). FDA-approved antagonists of CXCR4 and CCR5 inhibit Aβ-induced rods in both rodent and human neurons with effective concentrations for 50% rod reduction (EC50) of 1–10 nM. Remarkably, two D-amino acid receptor-active peptides (RAP-103 and RAP-310) inhibit Aβ-induced rods with an EC50 of ~1 pM in mouse neurons and ~0.1 pM in human neurons. These peptides are analogs of D-Ala-Peptide T-Amide (DAPTA) and share a pentapeptide sequence (TTNYT) antagonistic to several CCR-dependent responses. RAP-103 does not inhibit neuritogenesis or outgrowth even at 1 µM, >106-fold above its EC50. N-terminal methylation, or D-Thr to D-Ser substitution, decreases the rod-inhibiting potency of RAP-103 by 103-fold, suggesting high target specificity. Neither RAP peptide inhibits neuronal rod formation induced by excitotoxic glutamate, but both inhibit rods induced in human neurons by several PrPC/NOX pathway activators (Aβ, HIV-gp120 protein, and IL-6). Significantly, RAP-103 completely protects against Aβ-induced loss of mature and developing synapses and, at 0.1 nM, reverses rods in both rodent and human neurons (T½ ~ 3 h) even in the continuous presence of Aβ. Thus, this orally available, brain-permeable peptide should be highly effective in reducing rod pathology in multifactorial neurological diseases with mixed proteinopathies acting through PrPC/NOX.

Background Dysregulation of microRNA (miRNA) expression in the brain is a common feature of neurodegenerative diseases. Beyond their conventional role in regulating gene expression at the post-transcriptional level, certain miRNAs can act extracellularly as signaling molecules. Our study elucidates the identity of such miRNA species serving as ligands for membrane receptors expressed in central nervous system (CNS) neurons and the impact of such miRNAs on neurons in the context of neurodegenerative disease. Methods We combined a machine learning approach with the analysis of disease-associated miRNA databases to predict Alzheimer’s disease (AD)-associated miRNAs as potential signaling molecules for single-stranded RNA-sensing Toll-like receptors (TLRs) 7 and 8. TLR-expressing HEK-Blue reporter cells, primary murine microglia, and human THP-1 macrophages were used to validate the AD miRNAs as ligands for human and mouse TLR7 and/or TLR8. Interaction between mouse cortical neurons and extracellularly applied AD miRNAs was analyzed by live cell imaging and confocal microscopy. Transcriptome changes in cortical neurons exposed to AD miRNAs were assessed by RNAseq and RT-qPCR. The extracellular AD miRNAs’ effects on CNS neuron structure were investigated in cell cultures of murine primary cortical neurons and iPSC-derived human cortical neurons by immunocytochemistry. We employed a mouse model of intrathecal injection to assess effects of AD miRNAs acting as signaling molecules on neurons in vivo. Results We identified the AD-associated miRNAs miR-124-5p, miR-92a-1-5p, miR-9-5p, and miR-501-3p as novel endogenous ligands for TLR7 and/or TLR8. These miRNAs being extracellularly stable and active were taken up by murine cortical neurons via endocytosis and induced changes in neuronal inflammation-, proliferation-, and apoptosis-related gene expression. Exposure of both murine and human cortical neurons to the AD-associated miRNAs led to alterations of dendrite and axon structure, synapse protein expression, and cell viability in a sequence-dependent fashion. Extracellular introduction of the AD miRNAs into the cerebrospinal fluid of mice resulted in both changes in neuronal structure and synapses, and neuronal loss in the cerebral cortex. Most of the observed extracellular miRNA-induced effects on cortical neurons involved TLR7/8 signaling. Conclusion Neurodegenerative disease-associated miRNAs in extracellular form act as signaling molecules for CNS neurons including human cortical neurons, thereby modulating their structure and viability.

Background Motor neurons (MNs), which are primarily affected in amyotrophic lateral sclerosis (ALS), are a specialized type of neurons that are long and non-dividing. Given their unique structure, these cells heavily rely on transport of organelles along their axons and the process of autophagy to maintain their cellular homeostasis. It has been shown that disruption of the autophagy pathway is sufficient to cause progressive neurodegeneration and defects in autophagy have been associated with various subtypes of ALS, including those caused by hexanucleotide repeat expansions in the C9orf72 gene. A more comprehensive understanding of the dysfunctional cellular mechanisms will help rationalize the design of potent and selective therapies for C9orf72 -ALS. Methods In this study, we used induced pluripotent stem cell (iPSC)-derived MNs from C9orf72 -ALS patients and isogenic control lines to identify the underlying mechanisms causing dysregulations of the autophagy-lysosome pathway. Additionally, to ascertain the potential impact of C9orf72 loss-of-function on autophagic defects, we characterized the observed phenotypes in a C9orf72 knockout iPSC line (C9-KO). Results Despite the evident presence of dysfunctions in several aspects of the autophagy-lysosome pathway, such as disrupted lysosomal homeostasis, abnormal lysosome morphology, inhibition of autophagic flux, and accumulation of p62 in C9orf72 -ALS MNs, we were surprised to find that C9orf72 loss-of-function had minimal influence on these phenotypes. Instead, we primarily observed impairment in endosome maturation as a result of C9orf72 loss-of-function. Additionally, our study shed light on the pathological mechanisms underlying C9orf72 -ALS, as we detected an increased TBK1 phosphorylation at S172 in MNs derived from C9orf72 ALS patients. Conclusions Our data provides further insight into the involvement of defects in the autophagy-lysosome pathway in C9orf72 -ALS and strongly indicate that those defects are mainly due to the toxic gain-of-function mechanisms underlying C9orf72 -ALS.