Neuropeptide FF (NPFF)-positive nerve cells of the human cerebral cortex and white matter in controls, selected neurodegenerative diseases, and schizophrenia.

We quantified and determined for the first time the distribution pattern of the neuropeptide NPFF in the human cerebral cortex and subjacent white matter. To do so, we studied n = 9 cases without neurological disorders and n = 22 cases with neurodegenerative diseases, including sporadic amyotrophic lateral sclerosis (ALS, n = 8), Alzheimer's disease (AD, n = 8), Pick's disease (PiD, n = 3), and schizophrenia (n = 3). NPFF-immunopositive cells were located chiefly, but not exclusively, in the superficial white matter and constituted there a subpopulation of white matter interstitial cells (WMIC): Pyramidal-like and multipolar somata predominated in the gyral crowns, whereas bipolar and ovoid somata predominated in the cortex surrounding the sulci. Their sparsely ramified axons were unmyelinated and exhibited NPFF-positive bead-like varicosities. We found significantly fewer NPFF-immunopositive cells in the gray matter of the frontal, cingulate, and superior temporal gyri of both sporadic ALS and late-stage AD patients than in controls, and significantly fewer NPFF-positive cells in the subjacent as well as deep white matter of the frontal gyrus of these patients compared to controls. Notably, the number of NPFF-positive cells was also significantly lower in the hippocampal formation in AD compared to controls. In PiD, NPFF-positive cells were present in significantly lower numbers in the gray and white matter of the cingulate and frontal gyrii in comparison to controls. In schizophrenic patients, lower wNPFF cell counts in the neocortex were significant and global (cingulate, frontal, superior temporal gyrus, medial, and inferior gyri). The precise functions of NPFF-positive cells and their relationship to the superficial corticocortical white matter U-fibers are currently unknown. Here, NPFF immunohistochemistry and expression characterize a previously unrecognized population of cells in the human brain, thereby providing a new entry-point for investigating their physiological and pathophysiological roles.

Dynamics of synaptic damage in severe traumatic brain injury revealed by cerebrospinal fluid SNAP-25 and VILIP-1.

BACKGROUND: Biomarkers of neuronal, glial cells and inflammation in traumatic brain injury (TBI) are available but they do not specifically reflect the damage to synapses, which represent the bulk volume of the brain. Experimental models have demonstrated extensive involvement of synapses in acute TBI, but biomarkers of synaptic damage in human patients have not been explored. METHODS: Single-molecule array assays were used to measure synaptosomal-associated protein-25 (SNAP-25) and visinin-like protein 1 (VILIP-1) (along with neurofilament light chain (NFL), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillar acidic protein (GFAP), interleukin-6 (IL-6) and interleukin-8 (IL-8)) in ventricular cerebrospinal fluid (CSF) samples longitudinally acquired during the intensive care unit (ICU) stay of 42 patients with severe TBI or 22 uninjured controls. RESULTS: CSF levels of SNAP-25 and VILIP-1 are strongly elevated early after severe TBI and decline in the first few days. SNAP-25 and VILIP-1 correlate with inflammatory markers at two distinct timepoints (around D1 and then again at D5) in follow-up. SNAP-25 and VILIP-1 on the day-of-injury have better sensitivity and specificity for unfavourable outcome at 6 months than NFL, UCH-L1 or GFAP. Later elevation of SNAP-25 was associated with poorer outcome. CONCLUSION: Synaptic damage markers are acutely elevated in severe TBI and predict long-term outcomes, as well as, or better than, markers of neuroaxonal injury. Synaptic damage correlates with initial injury and with a later phase of secondary inflammatory injury.

Glycolic acid and D-lactate-putative products of DJ-1-restore neurodegeneration in FUS - and SOD1-ALS.

Amyotrophic lateral sclerosis (ALS) leads to death within 2-5 yr. Currently, available drugs only slightly prolong survival. We present novel insights into the pathophysiology of Superoxide Dismutase 1 (SOD1)- and in particular Fused In Sarcoma (FUS)-ALS by revealing a supposedly central role of glycolic acid (GA) and D-lactic acid (DL)-both putative products of the Parkinson's disease associated glyoxylase DJ-1. Combined, not single, treatment with GA/DL restored axonal organelle phenotypes of mitochondria and lysosomes in FUS- and SOD1-ALS patient-derived motoneurons (MNs). This was not only accompanied by restoration of mitochondrial membrane potential but even dependent on it. Despite presenting an axonal transport deficiency as well, TDP43 patient-derived MNs did not share mitochondrial depolarization and did not respond to GA/DL treatment. GA and DL also restored cytoplasmic mislocalization of FUS and FUS recruitment to DNA damage sites, recently reported being upstream of the mitochondrial phenotypes in FUS-ALS. Whereas these data point towards the necessity of individualized (gene-) specific therapy stratification, it also suggests common therapeutic targets across different neurodegenerative diseases characterized by mitochondrial depolarization.

Combined expansion and STED microscopy reveals altered fingerprints of postsynaptic nanostructure across brain regions in ASD-related SHANK3-deficiency.

Synaptic dysfunction is a key feature of SHANK-associated disorders such as autism spectrum disorder, schizophrenia, and Phelan-McDermid syndrome. Since detailed knowledge of their effect on synaptic nanostructure remains limited, we aimed to investigate such alterations in ex11|SH3 SHANK3-KO mice combining expansion and STED microscopy. This enabled high-resolution imaging of mosaic-like arrangements formed by synaptic proteins in both human and murine brain tissue. We found distinct shape-profiles as fingerprints of the murine postsynaptic scaffold across brain regions and genotypes, as well as alterations in the spatial and molecular organization of subsynaptic domains under SHANK3-deficient conditions. These results provide insights into synaptic nanostructure in situ and advance our understanding of molecular mechanisms underlying synaptic dysfunction in neuropsychiatric disorders.

Disrupted extracellular matrix and cell cycle genes in autism-associated Shank3 deficiency are targeted by lithium.

The Shank3 gene encodes the major postsynaptic scaffolding protein SHANK3. Its mutation causes a syndromic form of autism spectrum disorder (ASD): Phelan-McDermid Syndrome (PMDS). It is characterized by global developmental delay, intellectual disorders (ID), ASD behavior, affective symptoms, as well as extra-cerebral symptoms. Although Shank3 deficiency causes a variety of molecular alterations, they do not suffice to explain all clinical aspects of this heterogenic syndrome. Since global gene expression alterations in Shank3 deficiency remain inadequately studied, we explored the transcriptome in vitro in primary hippocampal cells from Shank3∆11(-/-) mice, under control and lithium (Li) treatment conditions, and confirmed the findings in vivo. The Shank3∆11(-/-) genotype affected the overall transcriptome. Remarkably, extracellular matrix (ECM) and cell cycle transcriptional programs were disrupted. Accordingly, in the hippocampi of adolescent Shank3∆11(-/-) mice we found proteins of the collagen family and core cell cycle proteins downregulated. In vitro Li treatment of Shank3∆11(-/-) cells had a rescue-like effect on the ECM and cell cycle gene sets. Reversed ECM gene sets were part of a network, regulated by common transcription factors (TF) such as cAMP responsive element binding protein 1 (CREB1) and ß-Catenin (CTNNB1), which are known downstream effectors of synaptic activity and targets of Li. These TFs were less abundant and/or hypo-phosphorylated in hippocampi of Shank3∆11(-/-) mice and could be rescued with Li in vitro and in vivo. Our investigations suggest the ECM compartment and cell cycle genes as new players in the pathophysiology of Shank3 deficiency, and imply involvement of transcriptional regulators, which can be modulated by Li. This work supports Li as potential drug in the management of PMDS symptoms, where a Phase III study is ongoing.

Expression profiles of the autism-related SHANK proteins in the human brain.

BACKGROUND: SHANKs are major scaffolding proteins at postsynaptic densities (PSDs) in the central nervous system. Mutations in all three family members have been associated with neurodevelopmental disorders such as autism spectrum disorders (ASDs). Despite the pathophysiological importance of SHANK2 and SHANK3 mutations in humans, research on the expression of these proteins is mostly based on rodent model organisms. RESULTS: In the present study, cellular and neuropil SHANK2 expression was analyzed by immunofluorescence (IF) staining of post mortem human brain tissue from four male individuals (19 brain regions). Mouse brains were analyzed in comparison to evaluate the degree of phylogenetic conservation. Furthermore, SHANK2 and SHANK3 isoform patterns were compared in human and mouse brain lysates. While isoform expression and subcellular distribution were largely conserved, differences in neuropil levels of SHANK2 were found by IF staining: Maximum expression was concordantly measured in the cerebellum; however, higher SHANK2 expression was detected in the human brainstem and thalamus when compared to mice. One of the lowest SHANK2 levels was found in the human amygdala, a moderately expressing region in mouse. Quantification of SHANK3 IF in mouse brains unveiled a distribution comparable to humans. CONCLUSIONS: In summary, these data show that the overall expression pattern of SHANK is largely conserved in defined brain regions; however, differences do exist, which need to be considered in the translation of rodent studies. The summarized expression patterns of SHANK2 and SHANK3 should serve as a reference for future studies.

Neurodegeneration or dysfunction in Phelan-McDermid syndrome? A multimodal approach with CSF and computational MRI.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a rare multisystem disease with global developmental delay and autistic features. Genetically, the disease is based on a heterozygous deletion of chromosome 22q13.3 with involvement of at least part of the SHANK3 gene or heterozygous pathogenic variants in SHANK3. Pathophysiologically, this syndrome has been regarded as a synaptopathy, but current data suggest an additional concept, since axonal functions of neurons are also impaired, thus, the specific pathophysiological processes in this disease are not yet fully understood. Since symptoms of the autism spectrum, regression, and stagnation in development occur, we investigated whether neuroinflammatory and neurodegenerative processes may also play a role. To this end, we analysed biomarkers in cerebrospinal fluid (CSF) and parameters from magnetic resonance imaging with high-resolution structural T1w volumetry and diffusion tensor imaging analysis in 19 Phelan-McDermid syndrome patients. RESULTS: CSF showed no inflammation but abnormalities in tau protein and amyloid-ß concentrations, however, with no typical biomarker pattern as in Alzheimer's disease. It could be demonstrated that these CSF changes were correlated with integrity losses of the fibres in the corticospinal tract as well as in the splenium and dorsal part of the cingulum. High CSF levels of tau protein were associated with loss of integrity of fibres in the corticospinal tract; lower levels of amyloid-ß were associated with decreasing integrity of fibre tracts of the splenium and posterior cingulate gyrus. Volumetric investigations showed global atrophy of the white matter, but not the grey matter, and particularly not in temporal or mesiotemporal regions, as is typical in later stages of Alzheimer's disease. CONCLUSIONS: In summary, alterations of neurodegenerative CSF markers in PMS individuals could be demonstrated which were correlated with structural connectivity losses of the corticospinal tract, the splenium, and the dorsal part of the cingulum, which can also be associated with typical clinical symptoms in these patients. These findings might represent a state of dysfunctional processes with ongoing degenerative and regenerative processes or a kind of accelerated aging. This study should foster further clinical diagnostics like tau- and amyloid-PET imaging as well as novel scientific approaches especially in basic research for further mechanistic proof.

Shank3 related muscular hypotonia is accompanied by increased intracellular calcium concentrations and ion channel dysregulation in striated muscle tissue.

Phelan-McDermid syndrome (PMS) is a syndromic form of Autism Spectrum Disorders (ASD) classified as a rare genetic neurodevelopmental disorder featuring global developmental delay, absent or delayed speech, ASD-like behaviour and neonatal skeletal muscle hypotonia. PMS is caused by a heterozygous deletion of the distal end of chromosome 22q13.3 or SHANK3 mutations. We analyzed striated muscles of newborn Shank3Δ11(-/-) animals and found a significant enlargement of the sarcoplasmic reticulum as previously seen in adult Shank3Δ11(-/-) mice, indicative of a Shank3-dependent and not compensatory mechanism for this structural alteration. We analyzed transcriptional differences by RNA-sequencing of muscle tissue of neonatal Shank3Δ11(-/-) mice and compared those to Shank3(+/+) controls. We found significant differences in gene expression of ion channels crucial for muscle contraction and for molecules involved in calcium ion regulation. In addition, calcium storage- [i.e., Calsequestrin (CSQ)], calcium secretion- and calcium-related signaling-proteins were found to be affected. By immunostainings and Western blot analyses we could confirm these findings both in Shank3Δ11(-/-) mice and PMS patient muscle tissue. Moreover, alterations could be induced in vitro by the selective downregulation of Shank3 in C2C12 myotubes. Our results emphasize that SHANK3 levels directly or indirectly regulate calcium homeostasis in a cell autonomous manner that might contribute to muscular hypotonia especially seen in the newborn.

Shank3 deletion in PV neurons is associated with abnormal behaviors and neuronal functions that are rescued by increasing GABAergic signaling.

BACKGROUND: Phelan-McDermid syndrome (PMS) is a neurodevelopmental disorder characterized by developmental delay, intellectual disability, and autistic-like behaviors and is primarily caused by haploinsufficiency of SHANK3 gene. Currently, there is no specific treatment for PMS, highlighting the need for a better understanding of SHANK3 functions and the underlying pathophysiological mechanisms in the brain. We hypothesize that SHANK3 haploinsufficiency may lead to alterations in the inhibitory system, which could be linked to the excitatory/inhibitory imbalance observed in models of autism spectrum disorder (ASD). Investigation of these neuropathological features may shed light on the pathogenesis of PMS and potential therapeutic interventions. METHODS: We recorded local field potentials and visual evoked responses in the visual cortex of Shank3∆11-/- mice. Then, to understand the impact of Shank3 in inhibitory neurons, we generated Pv-cre+/- Shank3Fl/Wt conditional mice, in which Shank3 was deleted in parvalbumin-positive neurons. We characterized the phenotype of this murine model and we compared this phenotype before and after ganaxolone administration. RESULTS: We found, in the primary visual cortex, an alteration of the gain control of Shank3 KO compared with Wt mice, indicating a deficit of inhibition on pyramidal neurons. This alteration was rescued after the potentiation of GABAA receptor activity by Midazolam. Behavioral analysis showed an impairment in grooming, memory, and motor coordination of Pv-cre+/- Shank3Fl/Wt compared with Pv-cre+/- Shank3Wt/Wt mice. These deficits were rescued with ganaxolone, a positive modulator of GABAA receptors. Furthermore, we demonstrated that treatment with ganaxolone also ameliorated evocative memory deficits and repetitive behavior of Shank3 KO mice. LIMITATIONS: Despite the significant findings of our study, some limitations remain. Firstly, the neurobiological mechanisms underlying the link between Shank3 deletion in PV neurons and behavioral alterations need further investigation. Additionally, the impact of Shank3 on other classes of inhibitory neurons requires further exploration. Finally, the pharmacological activity of ganaxolone needs further characterization to improve our understanding of its potential therapeutic effects. CONCLUSIONS: Our study provides evidence that Shank3 deletion leads to an alteration in inhibitory feedback on cortical pyramidal neurons, resulting in cortical hyperexcitability and ASD-like behavioral problems. Specifically, cell type-specific deletion of Shank3 in PV neurons was associated with these behavioral deficits. Our findings suggest that ganaxolone may be a potential pharmacological approach for treating PMS, as it was able to rescue the behavioral deficits in Shank3 KO mice. Overall, our study highlights the importance of investigating the role of inhibitory neurons and potential therapeutic interventions in neurodevelopmental disorders such as PMS.

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.

Live Cell Imaging of ATP Levels Reveals Metabolic Compartmentalization within Motoneurons and Early Metabolic Changes in FUS ALS Motoneurons.

Motoneurons are one of the most energy-demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultrastructure, transport, and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and the proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression is not fully understood yet. Here, we utilize hiPCS-derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in fused in sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons are accompanied by an overall upregulation of mitochondrial components and a significant increase in metabolic rates that correspond to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of diseased motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments, with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts the metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms.

Patterns of synaptic loss in human amyotrophic lateral sclerosis spinal cord: a clinicopathological study.

Amyotrophic Lateral Sclerosis (ALS) is mainly characterized by the degeneration of corticospinal neurons and spinal α-motoneurons; vulnerable cells display prominent pTDP-43 inclusions. Evidence gathered from genetics, murine models, and iPSC-derived neurons point to the early involvement of synapses in the disease course and their crucial role in the pathogenic cascade. However, pathology studies, with specimens from large post-mortem cohorts, mapping the pattern of synaptic disturbances over clinical and neuropathological hallmarks of disease progression, are currently not available. Thus, the appearance and progression of synaptic degeneration in human ALS patients are currently not known, preventing a full validation of the murine and in vitro models. Here, we investigated the loss of synaptophysin-positive terminals in cervical, thoracic, and lumbar spinal cord samples from a retrospective cohort of n = 33 ALS patients and n = 8 healthy controls, and we correlated the loss of synapses against clinicodemographic features and neuropathological ALS stage. We found that, although dorsal and intermediate spinal cord laminae do not lose synapses, ALS patients displayed a substantial but variable loss of synapses in the ventral horn of lumbar and cervical spinal cord. The amount of synaptic loss was predicted by disease duration, by the clinical site of onset, and by the loss of α-motoneurons, although not by the fraction of pTDP-43-immunopositive α-motoneurons. Taken together, our findings validate the synaptic pathology observed in other models and suggest that pathogenic pathways unfolding in the spinal microenvironment are critical to the progressive disassembly of local synaptic connectivity.

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.

CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue.

BACKGROUND: Post mortem human brain tissue is an essential resource to study cell types, connectivity as well as subcellular structures down to the molecular setup of the central nervous system especially with respect to the plethora of brain diseases. A key method is immunostaining with fluorescent dyes, which allows high-resolution imaging in three dimensions of multiple structures simultaneously. Although there are large collections of formalin-fixed brains, research is often limited because several conditions arise that complicate the use of human brain tissue for high-resolution fluorescence microscopy. RESULTS: In this study, we developed a clearing approach for immunofluorescence-based analysis of perfusion- and immersion-fixed post mortem human brain tissue, termed human Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging / Immunostaining / In situ hybridization-compatible Tissue-hYdrogel (hCLARITY). hCLARITY is optimized for specificity by reducing off-target labeling and yields very sensitive stainings in human brain sections allowing for super-resolution microscopy with unprecedented imaging of pre- and postsynaptic compartments. Moreover, hallmarks of Alzheimer's disease were preserved with hCLARITY, and importantly classical 3,3'-diaminobenzidine (DAB) or Nissl stainings are compatible with this protocol. hCLARITY is very versatile as demonstrated by the use of more than 30 well performing antibodies and allows for de- and subsequent re-staining of the same tissue section, which is important for multi-labeling approaches, e.g., in super-resolution microscopy. CONCLUSIONS: Taken together, hCLARITY enables research of the human brain with high sensitivity and down to sub-diffraction resolution. It therefore has enormous potential for the investigation of local morphological changes, e.g., in neurodegenerative diseases.

Excessive self-grooming, gene dysregulation and imbalance between the striosome and matrix compartments in the striatum of Shank3 mutant mice.

Autism is characterized by atypical social communication and stereotyped behaviors. Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are detected in 1-2% of patients with autism and intellectual disability, but the mechanisms underpinning the symptoms remain largely unknown. Here, we characterized the behavior of Shank3 Δ11/Δ11 mice from 3 to 12 months of age. We observed decreased locomotor activity, increased stereotyped self-grooming and modification of socio-sexual interaction compared to wild-type littermates. We then used RNAseq on four brain regions of the same animals to identify differentially expressed genes (DEGs). DEGs were identified mainly in the striatum and were associated with synaptic transmission (e.g., Grm2, Dlgap1), G-protein-signaling pathways (e.g., Gnal, Prkcg1, and Camk2g), as well as excitation/inhibition balance (e.g., Gad2). Downregulated and upregulated genes were enriched in the gene clusters of medium-sized spiny neurons expressing the dopamine 1 (D1-MSN) and the dopamine 2 receptor (D2-MSN), respectively. Several DEGs (Cnr1, Gnal, Gad2, and Drd4) were reported as striosome markers. By studying the distribution of the glutamate decarboxylase GAD65, encoded by Gad2, we showed that the striosome compartment of Shank3 Δ11/Δ11 mice was enlarged and displayed much higher expression of GAD65 compared to wild-type mice. Altogether, these results indicate altered gene expression in the striatum of Shank3-deficient mice and strongly suggest, for the first time, that the excessive self-grooming of these mice is related to an imbalance in the striatal striosome and matrix compartments.

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.