A TBK1 variant causes autophagolysosomal and motoneuron pathology without neuroinflammation in mice.

Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.

Propranolol reduces the accumulation of cytotoxic aggregates in C9orf72-ALS/FTD in vitro models.

Mutations in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathogenetic mechanisms linked to this gene are a direct consequence of an aberrant intronic expansion of a GGGGCC hexanucleotide located between the 1a and 1b non-coding exons, which can be transcribed to form cytotoxic RNA foci or even translated into aggregation-prone dipeptide repeat proteins. Importantly, the abnormal length of these repeats affects also the expression levels of C9orf72 itself, which suggests haploinsufficiency as additional pathomechanism. Thus, it appears that both toxic gain of function and loss of function are distinct but still coexistent features contributing to the insurgence of the disease in case of C9orf72 mutations. In this study, we aimed at identifying a strategy to address both aspects of the C9orf72-related pathobiochemistry and provide proof-of-principle information for a better understanding of the mechanisms leading to neuronal loss. By using primary neurons overexpressing toxic poly(GA), the most abundant protein product of the GGGGCC repeats, we found that the antiarrhythmic drug propranolol could efficiently reduce the accumulation of aberrant aggregates and increase the survival of C9orf72-related cultures. Interestingly, the improved catabolism appeared to not depend on major degradative pathways such as autophagy and the proteasome. By analyzing the proteome of poly(GA)-expressing neurons after exposure to propranolol, we found that the drug increased lysosomal degradation through a mechanism directly involving C9orf72 protein, whose levels were increased after treatment. Further confirmation of the beneficial effect of the beta blocker on aggregates' accumulation and survival of hiPSC-derived C9orf72-mutant motoneurons strengthened the finding that addressing both facets of C9orf72 pathology might represent a valid strategy for the treatment of these ALS/FTD cases.

Integrative proteomics highlight presynaptic alterations and c-Jun misactivation as convergent pathomechanisms in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease mainly affecting upper and lower motoneurons. Several functionally heterogeneous genes have been associated with the familial form of this disorder (fALS), depicting an extremely complex pathogenic landscape. This heterogeneity has limited the identification of an effective therapy, and this bleak prognosis will only improve with a greater understanding of convergent disease mechanisms. Recent evidence from human post-mortem material and diverse model systems has highlighted the synapse as a crucial structure actively involved in disease progression, suggesting that synaptic aberrations might represent a shared pathological feature across the ALS spectrum. To test this hypothesis, we performed the first comprehensive analysis of the synaptic proteome from post-mortem spinal cord and human iPSC-derived motoneurons carrying mutations in the major ALS genes. This integrated approach highlighted perturbations in the molecular machinery controlling vesicle release as a shared pathomechanism in ALS. Mechanistically, phosphoproteomic analysis linked the presynaptic vesicular phenotype to an accumulation of cytotoxic protein aggregates and to the pro-apoptotic activation of the transcription factor c-Jun, providing detailed insights into the shared pathobiochemistry in ALS. Notably, sub-chronic treatment of our iPSC-derived motoneurons with the fatty acid docosahexaenoic acid exerted a neuroprotective effect by efficiently rescuing the alterations revealed by our multidisciplinary approach. Together, this study provides strong evidence for the central and convergent role played by the synaptic microenvironment within the ALS spinal cord and highlights a potential therapeutic target that counteracts degeneration in a heterogeneous cohort of human motoneuron cultures.

Shank2 identifies a subset of glycinergic neurons involved in altered nociception in an autism model.

BACKGROUND: Autism Spectrum Disorders (ASD) patients experience disturbed nociception in the form of either hyposensitivity to pain or allodynia. A substantial amount of processing of somatosensory and nociceptive stimulus takes place in the dorsal spinal cord. However, many of these circuits are not very well understood in the context of nociceptive processing in ASD. METHODS: We have used a Shank2-/- mouse model, which displays a set of phenotypes reminiscent of ASD, and performed behavioural and microscopic analysis to investigate the role of dorsal horn circuitry in nociceptive processing of ASD. RESULTS: We determined that Shank2-/- mice display increased sensitivity to formalin pain and thermal preference, but a sensory specific mechanical allodynia. We demonstrate that high levels of Shank2 expression identifies a subpopulation of neurons in murine and human dorsal spinal cord, composed mainly by glycinergic interneurons and that loss of Shank2 causes the decrease in NMDAR in excitatory synapses on these inhibitory interneurons. In fact, in the subacute phase of the formalin test, glycinergic interneurons are strongly activated in wild type (WT) mice but not in Shank2-/- mice. Consequently, nociception projection neurons in laminae I are activated in larger numbers in Shank2-/- mice. LIMITATIONS: Our investigation is limited to male mice, in agreement with the higher representation of ASD in males; therefore, caution should be applied to extrapolate the findings to females. Furthermore, ASD is characterized by extensive genetic diversity and therefore the findings related to Shank2 mutant mice may not necessarily apply to patients with different gene mutations. Since nociceptive phenotypes in ASD range between hyper- and hypo-sensitivity, diverse mutations may affect the circuit in opposite ways. CONCLUSION: Our findings prove that Shank2 expression identifies a new subset of inhibitory interneurons involved in reducing the transmission of nociceptive stimuli and whose unchecked activation is associated with pain hypersensitivity. We provide evidence that dysfunction in spinal cord pain processing may contribute to the nociceptive phenotypes in ASD.

Multiomics and machine-learning identify novel transcriptional and mutational signatures in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal and incurable neurodegenerative disease that mainly affects the neurons of the motor system. Despite the increasing understanding of its genetic components, their biological meanings are still poorly understood. Indeed, it is still not clear to which extent the pathological features associated with amyotrophic lateral sclerosis are commonly shared by the different genes causally linked to this disorder. To address this point, we combined multiomics analysis covering the transcriptional, epigenetic and mutational aspects of heterogenous human induced pluripotent stem cell-derived C9orf72-, TARDBP-, SOD1- and FUS-mutant motor neurons as well as datasets from patients' biopsies. We identified a common signature, converging towards increased stress and synaptic abnormalities, which reflects a unifying transcriptional program in amyotrophic lateral sclerosis despite the specific profiles due to the underlying pathogenic gene. In addition, whole genome bisulphite sequencing linked the altered gene expression observed in mutant cells to their methylation profile, highlighting deep epigenetic alterations as part of the abnormal transcriptional signatures linked to amyotrophic lateral sclerosis. We then applied multi-layer deep machine-learning to integrate publicly available blood and spinal cord transcriptomes and found a statistically significant correlation between their top predictor gene sets, which were significantly enriched in toll-like receptor signalling. Notably, the overrepresentation of this biological term also correlated with the transcriptional signature identified in mutant human induced pluripotent stem cell-derived motor neurons, highlighting novel insights into amyotrophic lateral sclerosis marker genes in a tissue-independent manner. Finally, using whole genome sequencing in combination with deep learning, we generated the first mutational signature for amyotrophic lateral sclerosis and defined a specific genomic profile for this disease, which is significantly correlated to ageing signatures, hinting at age as a major player in amyotrophic lateral sclerosis. This work describes innovative methodological approaches for the identification of disease signatures through the combination of multiomics analysis and provides novel knowledge on the pathological convergencies defining amyotrophic lateral sclerosis.

Fast and efficient synaptosome isolation and post-synaptic density enrichment from hiPSC-motor neurons by biochemical sub-cellular fractionation.

We describe here a time-efficient, in-house protocol for synaptosome isolation and enrichment of the post-synaptic density (PSD) from hiPSC-derived motor neurons. By using biochemical sub-cellular fractionation, the crude synaptosome is first isolated from the cytosol and is then further separated into the synaptic cytosol and the enriched PSD fraction. The protocol can also potentially be adapted to other hiPSC-derived neuronal types, with necessary changes made to cell seeding density and buffer volumes.

Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Shank2/3 double knockout-based screening of cortical subregions links the retrosplenial area to the loss of social memory in autism spectrum disorders.

Members of the Shank protein family are master scaffolds of the postsynaptic architecture and mutations within the SHANK genes are causally associated with autism spectrum disorders (ASDs). We generated a Shank2-Shank3 double knockout mouse that is showing severe autism related core symptoms, as well as a broad spectrum of comorbidities. We exploited this animal model to identify cortical brain areas linked to specific autistic traits by locally deleting Shank2 and Shank3 simultaneously. Our screening of 10 cortical subregions revealed that a Shank2/3 deletion within the retrosplenial area severely impairs social memory, a core symptom of ASD. Notably, DREADD-mediated neuronal activation could rescue the social impairment triggered by Shank2/3 depletion. Data indicate that the retrosplenial area has to be added to the list of defined brain regions that contribute to the spectrum of behavioural alterations seen in ASDs.

Aging-Dependent Altered Transcriptional Programs Underlie Activity Impairments in Human C9orf72-Mutant Motor Neurons.

Amyotrophic Lateral Sclerosis (ALS) is an incurable neurodegenerative disease characterized by dysfunction and loss of upper and lower motor neurons (MN). Despite several studies identifying drastic alterations affecting synaptic composition and functionality in different experimental models, the specific contribution of impaired activity to the neurodegenerative processes observed in ALS-related MN remains controversial. In particular, contrasting lines of evidence have shown both hyper- as well as hypoexcitability as driving pathomechanisms characterizing this specific neuronal population. In this study, we combined high definition multielectrode array (HD-MEA) techniques with transcriptomic analysis to longitudinally monitor and untangle the activity-dependent alterations arising in human C9orf72-mutant MN. We found a time-dependent reduction of neuronal activity in ALSC9orf72 cultures occurring as synaptic contacts undergo maturation and matched by a significant loss of mutant MN upon aging. Notably, ALS-related neurons displayed reduced network synchronicity most pronounced at later stages of culture, suggesting synaptic imbalance. In concordance with the HD-MEA data, transcriptomic analysis revealed an early up-regulation of synaptic terms in ALSC9orf72 MN, whose expression was decreased in aged cultures. In addition, treatment of older mutant cells with Apamin, a K+ channel blocker previously shown to be neuroprotective in ALS, rescued the time-dependent loss of firing properties observed in ALSC9orf72 MN as well as the expression of maturity-related synaptic genes. All in all, this study broadens the understanding of how impaired synaptic activity contributes to MN degeneration in ALS by correlating electrophysiological alterations to aging-dependent transcriptional programs.

Motoneuron-Specific PTEN Deletion in Mice Induces Neuronal Hypertrophy and Also Regeneration after Facial Nerve Injury.

In postmitotic neurons, several tumor suppressor genes (TSGs), including p53, Rb, and PTEN, modulate the axon regeneration success after injury. Particularly, PTEN inhibition is a key driver of successful CNS axon regeneration after optic nerve or spinal cord injury. In contrast, in peripheral neurons, TSG influence in neuronal morphology, physiology, and pathology has not been investigated to the same depth. In this study, we conditionally deleted PTEN from mouse facial motoneurons (Chat-Cre/PtenloxP/loxP ) and analyzed neuronal responses in vivo with or without peripheral facial nerve injury in male and female mice. In uninjured motoneurons, PTEN loss induced somatic, axonal, and nerve hypertrophy, synaptic terminal enlargement and reduction in physiological whisker movement. Despite these morphologic and physiological changes, PTEN deletion positively regulated facial nerve regeneration and recovery of whisker movement after nerve injury. Regenerating PTEN-deficient motoneurons upregulated P-CREB and a signaling pathway involving P-Akt, P-PRAS40, P-mTOR, and P-4EBP1. In aged mice (12 months), PTEN deletion induced hair loss and facial hyperplasia of the epidermis. This suggests a time window in younger mice with PTEN loss stimulating axon growth after injury, however, at the risk of hyperplasia formation at later time points in the old animal. Overall, our data highlight a dual TSG function with PTEN loss impairing physiological neuron function but furthermore underscoring the positive effects of PTEN ablation in axon regeneration also for the PNS.SIGNIFICANCE STATEMENT Tumor suppressor genes (TSGs) restrict cell proliferation and growth. TSG inhibition, including p53 and PTEN, stimulates axon regeneration after CNS injury. In contrast, in PNS axon regeneration, TSGs have not been analyzed in great depth. Herein we show enhanced peripheral axon regeneration after PTEN deletion from facial motoneurons. This invokes a signaling cascade with novel PTEN partners, including CREB and PRAS40. In adult mice, PTEN loss induces hyperplasia of the skin epidermis, suggesting detrimental consequences when reaching adulthood in contrast to a beneficial TSG role for regeneration in young adult mice. Thus, our data highlight the double-edged sword nature of interfering with TSG function.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Synaptic disruption and CREB-regulated transcription are restored by K+ channel blockers in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, which is still missing effective therapeutic strategies. Although manipulation of neuronal excitability has been tested in murine and human ALS models, it is still under debate whether neuronal activity might represent a valid target for efficient therapies. In this study, we exploited a combination of transcriptomics, proteomics, optogenetics and pharmacological approaches to investigate the activity-related pathological features of iPSC-derived C9orf72-mutant motoneurons (MN). We found that human ALSC9orf72 MN are characterized by accumulation of aberrant aggresomes, reduced expression of synaptic genes, loss of synaptic contacts and a dynamic "malactivation" of the transcription factor CREB. A similar phenotype was also found in TBK1-mutant MN and upon overexpression of poly(GA) aggregates in primary neurons, indicating a strong convergence of pathological phenotypes on synaptic dysregulation. Notably, these alterations, along with neuronal survival, could be rescued by treating ALS-related neurons with the K+ channel blockers Apamin and XE991, which, respectively, target the SK and the Kv7 channels. Thus, our study shows that restoring the activity-dependent transcriptional programme and synaptic composition exerts a neuroprotective effect on ALS disease progression.

Corticotropin-releasing hormone (CRH) alters mitochondrial morphology and function by activating the NF-kB-DRP1 axis in hippocampal neurons.

Neuronal stress-adaptation combines multiple molecular responses. We have previously reported that thorax trauma induces a transient loss of hippocampal excitatory synapses mediated by the local release of the stress-related hormone corticotropin-releasing hormone (CRH). Since a physiological synaptic activity relies also on mitochondrial functionality, we investigated the direct involvement of mitochondria in the (mal)-adaptive changes induced by the activation of neuronal CRH receptors 1 (CRHR1). We observed, in vivo and in vitro, a significant shift of mitochondrial dynamics towards fission, which correlated with increased swollen mitochondria and aberrant cristae. These morphological changes, which are associated with increased NF-kB activity and nitric oxide concentrations, correlated with a pronounced reduction of mitochondrial activity. However, ATP availability was unaltered, suggesting that neurons maintain a physiological energy metabolism to preserve them from apoptosis under CRH exposure. Our findings demonstrate that stress-induced CRHR1 activation leads to strong, but reversible, modifications of mitochondrial dynamics and morphology. These alterations are accompanied by bioenergetic defects and the reduction of neuronal activity, which are linked to increased intracellular oxidative stress, and to the activation of the NF-kB/c-Abl/DRP1 axis.

Retinoic acid worsens ATG10-dependent autophagy impairment in TBK1-mutant hiPSC-derived motoneurons through SQSTM1/p62 accumulation.

Mutations in the TBK1 (TANK binding kinase 1) gene are causally linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TBK1 phosphorylates the cargo receptors OPTN and SQSTM1 regulating a critical step in macroautophagy/autophagy. Disruption of the autophagic flux leads to accumulation of cytosolic protein aggregates, which are a hallmark of ALS. hiPSC-derived TBK1-mutant motoneurons (MNs) showed reduced TBK1 levels and accumulation of cytosolic SQSTM1-positive aggresomes. By screening a library of nuclear-receptor-agonists for modifiers of the SQSTM1 aggregates, we identified 4-hydroxy(phenyl)retinamide (4HPR) as a potent modifier exerting detrimental effects on mutant-TBK1 motoneurons fitness exacerbating the autophagy overload. We have shown by TEM that TBK1-mutant motoneurons accumulate immature phagophores due a failure in the elongation phase, and 4HPR further worsens the burden of dysfunctional phagophores. 4HPR-increased toxicity was associated with the upregulation of SQSTM1 in a context of strongly reduced ATG10, while rescue of ATG10 levels abolished 4HPR toxicity. Finally, we showed that 4HPR leads to a downregulation of ATG10 and to an accumulation of SQSTM1+ aggresomes also in hiPSC-derived C9orf72-mutant motoneurons. Our data show that cultured human motoneurons harboring mutations in TBK1 gene display typical ALS features, like decreased viability and accumulation of cytosolic SQSTM1-positive aggresomes. The retinoid 4HPR appears a strong negative modifier of the fitness of TBK1 and C9orf72-mutant MNs, through a pathway converging on the mismatch of initiated autophagy and ATG10 levels. Thus, autophagy induction appears not to be a therapeutic strategy for ALS unless the specific underlying pathway alterations are properly addressed. Abbreviations: 4HPR: 4-hydroxy(phenyl)retinamide; AKT: AKT1 serine/threonine kinase 1; ALS: amyotrophic lateral sclerosis; ATG: autophagy related; AVs: autophagic vesicle; C9orf72: chromosome 9 open reading frame 72; CASP3: caspase 3; CHAT: choline O-acetyltransferase; CYCS: cytochrome c, somatic; DIV: day in vitro; FTD: frontotemporal dementia; FUS: FUS RNA binding protein; GFP: green fluorescent protein; hiPSCs: human induced pluripotent stem cells; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MNs: motoneurons; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; RARA: retinoic acid receptor alpha; SLC18A3/VACHT: solute carrier family 18 (vesicular acetylcholine transporter), member 3; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TEM: transmission electron microscopy.

STEM tomography of high-pressure frozen and freeze-substituted cells: a comparison of image stacks obtained at 200 kV or 300 kV.

Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam ("parallel" beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip.

Nutrient limitation affects presynaptic structures through dissociable Bassoon autophagic degradation and impaired vesicle release.

Acute mismatch between metabolic requirements of neurons and nutrients/growth factors availability characterizes several neurological conditions such as traumatic brain injury, stroke and hypoglycemia. Although the effects of this mismatch have been investigated at cell biological level, the effects on synaptic structure and function are less clear. Since synaptic activity is the most energy-demanding neuronal function and it is directly linked to neuronal networks functionality, we have explored whether nutrient limitation (NL) affects the ultrastructure, function and composition of pre and postsynaptic terminals. We show that upon NL, presynaptic terminals show disorganized vesicle pools and reduced levels of the active zone protein Bassoon (but not of Piccolo). Moreover, NL triggers an impaired vesicle release, which is reversed by re-administration of glucose but not by the blockade of autophagic or proteasomal protein degradation. This reveals a dissociable correlation between presynaptic architecture and vesicle release, since restoring vesicle fusion does not necessarily depend from the rescue of Bassoon levels. Thus, our data show that the presynaptic compartment is highly sensitive to NL and the rescue of presynaptic function requires re-establishment of the metabolic supply rather than preventing local protein degradation.

NEK1 loss-of-function mutation induces DNA damage accumulation in ALS patient-derived motoneurons.

Mutations in genes coding for proteins involved in DNA damage response (DDR) and repair, such as C9orf72 and FUS (Fused in Sarcoma), are associated with neurodegenerative diseases and lead to amyotrophic lateral sclerosis (ALS). Heterozygous loss-of-function mutations in NEK1 (NIMA-related kinase 1) have also been recently found to cause ALS. NEK1 codes for a multifunctional protein, crucially involved in mitotic checkpoint control and DDR. To resolve pathological alterations associated with NEK1 mutation, we compared hiPSC-derived motoneurons carrying a NEK1 mutation with mutant C9orf72 and wild type neurons at basal level and after DNA damage induction. Motoneurons carrying a C9orf72 mutation exhibited cell specific signs of increased DNA damage. This phenotype was even more severe in NEK1c.2434A>T neurons that showed significantly increased DNA damage at basal level and impaired DDR after induction of DNA damage in an maturation-dependent manner. Our results provide first mechanistic insight in pathophysiological alterations induced by NEK1 mutations and point to a converging pathomechanism of different gene mutations causative for ALS. Therefore, our study contributes to the development of novel therapeutic strategies to reduce DNA damage accumulation in neurodegenerative diseases and ALS.