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Adriaenssens et al. provide evidence suggesting that cytosolic small heat shock proteins localize to the mitochondrial intermembrane space, where they operate as molecular chaperones. Mitochondria are complex organelles with different compartments, each harbouring their own protein quality control factors. While chaperones of the mitochondrial matrix are well characterized, it is poorly understood which chaperones protect the mitochondrial intermembrane space. Here we show that cytosolic small heat shock proteins are imported under basal conditions into the mitochondrial intermembrane space, where they operate as molecular chaperones. Protein misfolding in the mitochondrial intermembrane space leads to increased recruitment of small heat shock proteins. Depletion of small heat shock proteins leads to mitochondrial swelling and reduced respiration, while aggregation of aggregation-prone substrates is countered in their presence. Charcot-Marie-Tooth disease-causing mutations disturb the mitochondrial function of HSPB1, potentially linking previously observed mitochondrial dysfunction in Charcot-Marie-Tooth type 2F to its role in the mitochondrial intermembrane space. Our results reveal that small heat shock proteins form a chaperone system that operates in the mitochondrial intermembrane space.

PURPOSE CTR9 is a subunit of the PAF1 complex (PAF1C) that plays a crucial role in transcription regulation by binding CTR9 to RNA polymerase II. It is involved in transcription-coupled histone modification through promoting H3K4 and H3K36 methylation. We describe the clinical and molecular studies in 13 probands, harboring likely pathogenic CTR9 missense variants, collected through GeneMatcher. METHODS Exome sequencing was performed in all individuals. CTR9 variants were assessed through 3-dimensional modeling of the activated human transcription complex Pol II-DSIF-PAF-SPT6 and the PAF1/CTR9 complex. H3K4/H3K36 methylation analysis, mitophagy assessment based on tetramethylrhodamine ethyl ester perchlorate immunofluorescence, and RNA-sequencing in skin fibroblasts from 4 patients was performed. RESULTS Common clinical findings were variable degrees of intellectual disability, hypotonia, joint hyperlaxity, speech delay, coordination problems, tremor, and autism spectrum disorder. Mild dysmorphism and cardiac anomalies were less frequent. For 11 CTR9 variants, de novo occurrence was shown. Three-dimensional modeling predicted a likely disruptive effect of the variants on local CTR9 structure and protein interaction. Additional studies in fibroblasts did not unveil the downstream functional consequences of the identified variants. CONCLUSION We describe a neurodevelopmental disorder caused by (mainly) de novo variants in CTR9, likely affecting PAF1C function.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2., See Müller and Horvath (doi:10.1093/brain/awab278) for a scientific commentary on this article. Van Lent et al. use neurons differentiated from patient-derived iPSCs to identify common hallmarks of axonal degeneration, including impaired axonal transport and mitochondrial dysfunction, shared by different CMT2 subtypes. Targeting these pathomechanisms could provide new drug development opportunities for CMT2.

Charcot-Marie-Tooth (CMT) disease is the most common inherited disorder of the peripheral nervous system. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the peripheral nervous system. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proven to be particularly challenging. Here, we present an iPSC-derived organoid culture, containing various cell types of the peripheral nervous system, including myelinating human Schwann cells, which mimics the human peripheral nervous system. Single-cell analysis confirmed the peripheral nervous system-like cellular composition and provides insight into the developmental trajectory. We used this organoid-model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion bulb-like formations in a later developmental stage. These hallmarks were not present in the for CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol, was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model is able to capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model to study demyelinating diseases, and supports the therapeutic approach of reducing PMP22 expression.

Dementia with Lewy bodies (DLB) and Parkinson’s disease (PD) are clinically, pathologically and etiologically disorders embedded in the Lewy body disease (LBD) continuum, characterized by neuronal α-synuclein pathology. Rare homozygous and compound heterozygous premature termination codon (PTC) mutations in the Vacuolar Protein Sorting 13 homolog C gene (VPS13C) are associated with early-onset recessive PD. We observed in two siblings with early-onset age (VPS13C, p.Trp395Cys and p.Ala444Pro, inherited from their healthy parents in a recessive manner. In lymphoblast cells of the index patient, the missense mutations reduced VPS13C expression by 90% (p = 0.0002). Subsequent, we performed targeted resequencing of VPS13C in 844 LBD patients and 664 control persons. Using the optimized sequence kernel association test, we obtained a significant association (p = 0.0233) of rare VPS13C genetic variants (minor allele frequency ≤ 1%) with LBD. Among the LBD patients, we identified one patient with homozygous missense mutations and three with compound heterozygous missense mutations in trans position, indicative for recessive inheritance. In four patients with compound heterozygous mutations, we were unable to determine trans position. The frequency of LBD patient carriers of proven recessive compound heterozygous missense mutations is 0.59% (5/844). In autopsy brain tissue of two unrelated LBD patients, the recessive compound heterozygous missense mutations reduced VPS13C expression. Overexpressing of wild type or mutant VPS13C in HeLa or SH-SY5Y cells, demonstrated that the mutations p.Trp395Cys or p.Ala444Pro, abolish the endosomal/lysosomal localization of VPS13C. Overall, our data indicate that rare missense mutations in VPS13C are associated with LBD and recessive compound heterozygous missense mutations might have variable effects on the expression and functioning of VPS13C. We conclude that comparable to the recessive inherited PTC mutations in VPS13C, combinations of rare recessive compound heterozygous missense mutations reduce VPS13C expression and contribute to increased risk of LBD.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot‐Marie‐Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C‐terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient‐derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V‐containing proteins. We identified multiple IxI/V‐bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V‐binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein–protein interactions., NMR studies define how the P182L mutation alters intramolecular interactions to increase HSP27 accessibility for numerous binding proteins, compromising its chaperone function in patient cell‐derived motor neurons.

Summary Most neurological disorders display impaired synaptic connectivity. Hence, modulation of synapse formation may have therapeutic relevance. However, the high density and small size of synapses complicate their quantification. To improve synapse-oriented screens, we analyzed the labeling performance of synapse-targeting antibodies on neuronal cell cultures using segmentation-independent image analysis based on sliding window correlation. When assessing pairwise colocalization, a common readout for mature synapses, overlap was incomplete and confounded by spurious signals. To circumvent this, we implemented a proximity ligation-based approach that only leads to a signal when two markers are sufficiently close. We applied this approach to different marker combinations and demonstrate its utility for detecting synapse density changes in healthy and compromised cultures. Thus, segmentation-independent analysis and exploitation of resident protein proximity increases the sensitivity of synapse quantifications in neuronal cultures and represents a valuable extension to the analytical toolset for in vitro synapse screens., Graphical Abstract, Highlights • Synapse antibody library screen using segmentation-independent image analysis • Colocalization of pre- and postsynaptic markers partially reveals mature synapses • Transsynaptic proximity ligation assay enhances sensitivity • Guidelines for synapse-oriented in vitro screening assays, Optical Imaging; Molecular Biology Experimental Approach; Cellular Neuroscience ; Biocomputational Method

Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.

Background Dementia with Lewy bodies (DLB) and Parkinson’s disease (PD) are clinically, pathologically and etiologically overlapping disorders. They are included in the Lewy body disease (LBD) continuum characterized by α-synuclein-positive Lewy body pathology in neurons. Homozygous PTC mutations in Vacuolar Protein Sorting 13 homolog C gene (VPS13C) are associated with early-onset PD. Methods Whole genome sequencing of two affected siblings and healthy parents of family A with onset age below 50 years and DLB pathology confirmed at autopsy. Targeted resequencing of the VPS13C coding region in 844 LBD patients and 664 control individuals. Phasing cis-trans location of the compound heterozygous VPS13C variants. Analysis of VPS13C protein expression in lymphoblast cells and brain lysates. Immunohistochemistry and live cell labeling in HeLa and SH-SY5Y cells overexpressing wild type or mutant VPS13C. Results In the two affected siblings of family A, we identified compound heterozygous rare variants located in trans in VPS13C Rare VPS13C variants (MAF ≤ 1%), are significantly associated (p = 0.0233) in the LBD patient cohort using optimized sequence kernel association test (SKAT-O). We identified 11 carriers of compound heterozygous variants and 1 carrier of homozygous variants in VPS13C. Trans location of the variants in compound heterozygous carriers was confirmed in 4 and cis location in 3 patients. The frequency of patient carriers with bi-allelic variants mimicking recessive inheritance is 1.07% (9/844). In post-mortem brain tissue of two unrelated DLB carriers of compound trans heterozygous variants, we observed a reduction of VPS13C protein expression. Overexpressing of wild type or mutant VPS13C, in HeLa and SH-SY5Y cells, demonstrated that the mutations abolish the endosomal/lysosomal localization of VPS13C. Conclusions Our data indicate that rare alleles are associated with LBD, and when present bi-allelic in patients, these combinations have variable effects on expression and functioning of VPS13C. Apart from the recessive PTC mutations, some combinations of rare missense mutations might mimic recessive inheritance and explain part of the sporadic LBD patients. We propose that homozygous or compound heterozygous rare missense variants in VPS13C reducing VPS13C protein expression can contribute to risk of LBD.

The authors identify two subsets of peripheral nerve macrophages residing in the endoneurium and the epineurium and displaying a distinct transcriptome and response to injury. These cells lack the main microglia identity and have a distinct origin. While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relm alpha(+)Mgl1(+) snMacs in the epineurium and Relm alpha(-)Mgl1(-) snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

Human molecular genetics: HMG online 28(4), 615-627 (2019). doi:10.1093/hmg/ddy352, Published by Oxford Univ. Press, Oxford

Distal hereditary motor neuropathies are a rare subgroup of inherited peripheral neuropathies hallmarked by a length-dependent axonal degeneration of lower motor neurons without significant involvement of sensory neurons. We identified patients with heterozygous nonsense mutations in the αII-spectrin gene, SPTAN1, in three separate dominant hereditary motor neuropathy families via next-generation sequencing. Variable penetrance was noted for these mutations in two of three families, and phenotype severity differs greatly between patients. The mutant mRNA containing nonsense mutations is broken down by nonsense-mediated decay and leads to reduced protein levels in patient cells. Previously, dominant-negative αII-spectrin gene mutations were described as causal in a spectrum of epilepsy phenotypes.

ObjectiveTo study the genetic and phenotypic spectrum of patients harboring recessive mutations in BVES.MethodsWe performed whole-exome sequencing in a multicenter cohort of 1929 patients with a suspected hereditary myopathy, showing unexplained limb-girdle muscular weakness and/or elevated creatine kinase levels. Immunohistochemistry and mRNA experiments on patients' skeletal muscle tissue were performed to study the pathogenicity of identified loss-of-function (LOF) variants in BVES.ResultsWe identified 4 individuals from 3 families harboring homozygous LOF variants in BVES, the gene that encodes for Popeye domain containing protein 1 (POPDC1). Patients showed skeletal muscle involvement and cardiac conduction abnormalities of varying nature and severity, but all exhibited at least subclinical signs of both skeletal muscle and cardiac disease. All identified mutations lead to a partial or complete loss of function of BVES through nonsense-mediated decay or through functional changes to the POPDC1 protein.ConclusionsWe report the identification of homozygous LOF mutations in BVES, causal in a young adult-onset myopathy with concomitant cardiac conduction disorders in the absence of structural heart disease. These findings underline the role of POPDC1, and by extension, other members of this protein family, in striated muscle physiology and disease. This disorder appears to have a low prevalence, although it is probably underdiagnosed because of its striking phenotypic variability and often subtle yet clinically relevant manifestations, particularly concerning the cardiac conduction abnormalities.

Acta neuropathologica 135(1), 131-148 (2018). doi:10.1007/s00401-017-1756-0, Published by Springer, Berlin ; Heidelberg

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects.

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.

SUMMARYThe ER is a complex network of sheets and tubules that is continuously being remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in Atlastins (ATL), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is largely unknown. Here we show by EM volume reconstruction of transfected cells, neurons and patient fibroblasts that the HSAN-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity, but are unable to promote membrane fusion, suggesting a defect on an intermediate step of the ATL3 functional cycle. Our data therefore show that the effects of ATL3 mutations on ER network organization stretch beyond a loss of fusion, shedding a new light on neuropathies caused by atlastin defects.

Objective:To identify the unknown genetic cause in a nuclear family with an axonal form of peripheral neuropathy and atypical disease course.Methods:Detailed neurologic, electrophysiologic, and neuropathologic examinations of the patients were performed. Whole exome sequencing of both affected individuals was done. The effect of the identified sequence variations was investigated at cDNA and protein level in patient-derived lymphoblasts. The plasma sphingoid base profile was analyzed. Functional consequences of neuron-specific downregulation of the gene were studied in Drosophila.Results:Both patients present an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. We identified compound heterozygous mutations cosegregating with disease and absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles.Conclusions:We suggest SPL deficiency as a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. Our data emphasize the importance of sphingolipid metabolism for neuronal function.

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders characterized by progressive spasticity of the lower limbs due to degeneration of the corticospinal motor neurons. In a Bulgarian family with three siblings affected by complicated hereditary spastic paraplegia, we performed whole exome sequencing and homozygosity mapping and identified a homozygous p.Thr512Ile (c.1535C>T) mutation in ATP13A2. Molecular defects in this gene have been causally associated with Kufor-Rakeb syndrome (#606693), an autosomal recessive form of juvenile-onset parkinsonism, and neuronal ceroid lipofuscinosis (#606693), a neurodegenerative disorder characterized by the intracellular accumulation of autofluorescent lipopigments. Further analysis of 795 index cases with hereditary spastic paraplegia and related disorders revealed two additional families carrying truncating biallelic mutations in ATP13A2. ATP13A2 is a lysosomal P5-type transport ATPase, the activity of which critically depends on catalytic autophosphorylation. Our biochemical and immunocytochemical experiments in COS-1 and HeLa cells and patient-derived fibroblasts demonstrated that the hereditary spastic paraplegia-associated mutations, similarly to the ones causing Kufor-Rakeb syndrome and neuronal ceroid lipofuscinosis, cause loss of ATP13A2 function due to transcript or protein instability and abnormal intracellular localization of the mutant proteins, ultimately impairing the lysosomal and mitochondrial function. Moreover, we provide the first biochemical evidence that disease-causing mutations can affect the catalytic autophosphorylation activity of ATP13A2. Our study adds complicated hereditary spastic paraplegia (SPG78) to the clinical continuum of ATP13A2-associated neurological disorders, which are commonly hallmarked by lysosomal and mitochondrial dysfunction. The disease presentation in our patients with hereditary spastic paraplegia was dominated by an adult-onset lower-limb predominant spastic paraparesis. Cognitive impairment was present in most of the cases and ranged from very mild deficits to advanced dementia with frontotemporal characteristics. Nerve conduction studies revealed involvement of the peripheral motor and sensory nerves. Only one of five patients with hereditary spastic paraplegia showed clinical indication of extrapyramidal involvement in the form of subtle bradykinesia and slight resting tremor. Neuroimaging cranial investigations revealed pronounced vermian and hemispheric cerebellar atrophy. Notably, reduced striatal dopamine was apparent in the brain of one of the patients, who had no clinical signs or symptoms of extrapyramidal involvement.

Charcot–Marie–Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.

The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.

Mutations in the small heat shock protein HSPB1 (HSP27) are a cause of axonal Charcot–Marie–Tooth neuropathy (CMT2F) and distal hereditary motor neuropathy. To better understand the effect of mutations in HSPB1 on the neuronal cytoskeleton, we stably transduced neuronal cells with wild-type and mutant HSPB1 and investigated axonal transport of neurofilaments (NFs). We observed that mutant HSPB1 affected the binding of NFs to the anterograde motor protein kinesin, reducing anterograde transport of NFs. These deficits were associated with an increased phosphorylation of NFs and cyclin-dependent kinase Cdk5. As Cdk5 mediates NF phosphorylation, inhibition of Cdk5/p35 restored NF phosphorylation level, as well as NF binding to kinesin in mutant HSPB1 neuronal cells. Altogether, we demonstrate that HSPB1 mutations induce hyperphosphorylation of NFs through Cdk5 and reduce anterograde transport of NFs. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1133-6) contains supplementary material, which is available to authorized users.

Purpose Amyloid deposition in the brain is considered an initial event in the progression of Alzheimer's disease. We hypothesized that the presence of amyloid plaques in the brain of APP/presenilin 1 mice leads to higher diffusion kurtosis measures due to increased microstructural complexity. As such, our purpose was to provide an in vivo proof of principle for detection of amyloidosis by diffusion kurtosis imaging (DKI). Methods APPKM670/671NL/presenilin 1 L166P mice (n = 5) and wild-type littermates (n = 5) underwent DKI at the age of 16 months. Averaged diffusion and diffusion kurtosis parameters were obtained for multiple regions (hippocampus–cortex–thalamus–cerebellum). After DKI, mice were sacrificed for amyloid staining. Results Histograms of the frequency distribution of the DKI parameters tended to shift to higher values. After normalization of absolute values to the cerebellum, a nearly plaque-free region, mean, radial, and axial diffusion kurtosis were significantly higher in APP/presenilin 1 mice as compared to wild-type in the cortex and thalamus, regions demonstrating substantial amyloid staining. Conclusion The current study, although small-scale, suggests increased DKI metrics, in the absence of alterations in diffusion tensor imaging metrics in the cortex and thalamus of APP/presenilin 1 mice with established amyloidosis. These results warrant further investigations on the potential of DKI as a sensitive marker for Alzheimer's disease. Magn Reson Med 69:1115–1121, 2013. © 2013 Wiley Periodicals, Inc.

Missense mutations in the small heat shock protein HSPB8 cause distal hereditary motor neuropathy (dHMN) and axonal Charcot-Marie-Tooth disease (CMT2L). We previously demonstrated that, despite the ubiquitous expression of HSPB8, motor neurons appear to be predominantly affected by HSPB8 mutations. Here, we studied the effect of mutant HSPB8 in primary fibroblast cultures derived from dHMN patients’ skin biopsy. In early passage cultures, we observed in all patients’ fibroblasts HSPB8 protein aggregates that were not detected in control cells. After applying heat shock stress on the patients’ early passage cultured cells, the protein aggregates coalesced into larger formations, while in control cells a homogenous upregulation of HSPB8 protein expression was seen. We also found a reduction in the mitochondrial membrane potential in the early passage cultures. After three months in culture, the number of cells with aggregates had become indistinguishable from that in controls and the mitochondrial membrane potential had returned to normal. These results emphasize the possible drawbacks of using patients’ non-neuronal cells to study neuropathological disease mechanisms.

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.

Neutrophil cell death plays a crucial role in neutrophil homeostasis and the resolution of inflammation. The superoxide-producing NADPH oxidase is involved in pathogen degradation and subsequent activation of cell death programs. Neutrophils from patients with chronic granulomatous disease, who have a deficient NADPH oxidase activity, have been demonstrated previously to have a prolonged lifespan, suggesting that a basal NADPH oxidase activity also regulates spontaneous neutrophil turnover. The NADPH oxidase inhibitor parabutoporin (PP) does delay spontaneous apoptosis, but this effect is completely independent of NADPH oxidase inhibition. Instead, the prosurvival effect of PP depends on activation of protein kinase B/Akt via lipid raft signaling. Disruption of lipid rafts abrogates the prosurvival effect without interfering with NADPH oxidase activity. Furthermore, we cannot detect a different rate of spontaneous apoptosis between normal and NADPH oxidase-deficient neutrophils, arguing against a role of NADPH oxidase in spontaneous neutrophil apoptosis.

In Arabidopsis, abscisic acid (ABA) application can induce resistance by priming for callose deposition; this resistance is impaired in ABA-deficient and -insensitive mutants. In tomato, ABA-deficiency causes resistance to Botrytis cinerea. Here, we show that callose deposition after B. cinerea inoculation is weaker in the ABA-deficient sitiens tomato mutant compared to the wild type (WT). Inhibition of callose synthesis did not affect resistance in sitiens, but caused additional susceptibility in WT. These findings indicate that callose deposition is not part of sitiens defence responses that are effective in blocking B. cinerea and suggest that callose deposition only contributes to WT basal resistance. Furthermore, also in tomato callose formation is at least in part ABA-dependent. However, it seems that in contrast to Arabidopsis, basal ABA levels in tomato are sufficiently high to prime for callose deposition.

Plant defense mechanisms against necrotrophic pathogens, such as Botrytis cinerea, are considered to be complex and to differ from those that are effective against biotrophs. In the abscisic acid-deficient sitiens tomato (Solanum lycopersicum) mutant, which is highly resistant to B. cinerea, accumulation of hydrogen peroxide (H2O2) was earlier and stronger than in the susceptible wild type at the site of infection. In sitiens, H2O2 accumulation was observed from 4 h postinoculation (hpi), specifically in the leaf epidermal cell walls, where it caused modification by protein cross-linking and incorporation of phenolic compounds. In wild-type tomato plants, H2O2 started to accumulate 24 hpi in the mesophyll layer and was associated with spreading cell death. Transcript-profiling analysis using TOM1 microarrays revealed that defense-related transcript accumulation prior to infection was higher in sitiens than in wild type. Moreover, further elevation of sitiens defense gene expression was stronger than in wild type 8 hpi both in number of genes and in their expression levels and confirmed a role for cell wall modification in the resistant reaction. Although, in general, plant defense-related reactive oxygen species formation facilitates necrotrophic colonization, these data indicate that timely hyperinduction of H2O2-dependent defenses in the epidermal cell wall can effectively block early development of B. cinerea.

Background The clusterin (CLU) gene has been identified as an important risk locus for Alzheimer’s disease (AD). Although the actual risk–increasing polymorphisms at this locus remain to be identified, we previously observed an increased frequency of rare non-synonymous mutations and small insertion-deletions of CLU in AD patients, which specifically clustered in the β-chain domain of CLU. Nonetheless the pathogenic nature of these variants remained unclear. Here we report a novel non-synonymous CLU mutation (p.I360N) in a Belgian Alzheimer patient and have explored the pathogenic nature of this and 10 additional CLU mutations on protein localization and secretion in vitro using immunocytochemistry, immunodetection and ELISAs. Results Three patient-specific CLU mutations in the β-chain (p.I303NfsX13, p.R338W and p.I360N) caused an alteration of the subcellular CLU localization and diminished CLU transport through the secretory pathway, indicative of possible degradation mechanisms. For these mutations, significantly reduced CLU intensity was observed in the Golgi while almost all CLU protein was exclusively present in the endoplasmic reticulum. This was further confirmed by diminished CLU secretion in HEK293T and HEK293 FLp-In cell lines. Conclusions Our data lend further support to the contribution of rare coding CLU mutations in the pathogenesis of neurodegenerative diseases. Functional analyses suggest reduced secretion of the CLU protein as the mode of action for three of the examined CLU mutations. One of those is a frameshift mutation leading to a loss of secreted protein, and the other two mutations are amino acid substitutions in the disulfide bridge region, possibly interfering with heterodimerization of the α- and β-chain of CLU. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0024-9) contains supplementary material, which is available to authorized users.

Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for the viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth (CMT) disease. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YAKS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies. (C) 2014 Elsevier Inc. All rights reserved.

The remodeling capacity of microtubules (MT) is essential for their proper function. In mammals, MTs are predominantly formed at the centrosome, but can also originate from non-centrosomal sites, a process that is still poorly understood. We here show that the small heat shock protein HSPB1 plays a role in the control of non-centrosomal MT formation. The HSPB1 expression level regulates the balance between centrosomal and non-centrosomal MTs. The HSPB1 protein can be detected specifically at sites of de novo forming non-centrosomal MTs, while it is absent from the centrosomes. In addition, we show that HSPB1 binds preferentially to the lattice of newly formed MTs in vitro, suggesting that its function occurs by stabilizing MT seeds. Our findings open new avenues for the understanding of the role of HSPB1 in the development, maintenance and protection of cells with specialized non-centrosomal MT arrays.

Background The activation of the immune system in neurodegeneration has detrimental as well as beneficial effects. Which aspects of this immune response aggravate the neurodegenerative breakdown and which stimulate regeneration remains an open question. To unravel the neuroprotective aspects of the immune system we focused on a model of acute peripheral nerve injury, in which the immune system was shown to be protective. Methods To determine the type of immune response triggered after axotomy of the sciatic nerve, a model for Wallerian degeneration in the peripheral nervous system, we evaluated markers representing the two extremes of a type I and type II immune response (classical vs. alternative) using real-time quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemistry. Results Our results showed that acute peripheral nerve injury triggers an anti-inflammatory and immunosuppressive response, rather than a pro-inflammatory response. This was reflected by the complete absence of classical macrophage markers (iNOS, IFNγ, and IL12p40), and the strong up-regulation of tissue repair markers (arginase-1, Ym1, and Trem2). The signal favoring the alternative macrophage environment was induced immediately after nerve damage and appeared to be established within the nerve, well before the infiltration of macrophages. In addition, negative regulators of the innate immune response, as well as the anti-inflammatory cytokine IL-10 were induced. The strict regulation of the immune system dampens the potential tissue damaging effects of an over-activated response. Conclusions We here demonstrate that acute peripheral nerve injury triggers an inherent protective environment by inducing the M2 phenotype of macrophages and the expression of arginase-1. We believe that the M2 phenotype, associated with a sterile inflammatory response and tissue repair, might explain their neuroprotective capacity. As such, shifting the neurodegeneration-induced immune responses towards an M2/Th2 response could be an important therapeutic strategy.

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.

International audience; Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy leading to progressive distal sensory loss and severe ulcerations. Mutations in SPTLC1 and SPTLC2, encoding the two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the first and rate-limiting step in the de novo synthesis of sphingolipids, have been reported to cause HSAN-I. Here, we demonstrate that the SPTLC1 mutations p.S331F and p.A352V result in a reduction of SPT activity in vitro and are associated with increased levels of the deoxysphingoid bases 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine in patients' plasma samples. Stably expressing p.S331F-SPTLC1 HEK293T cell lines likewise show accumulation of deoxysphingoid bases, but this accumulation is not observed in HEK293T cells overexpressing p.A352V-SPTLC1. These results confirm that the increased formation of deoxysphingoid bases is a key feature for HSAN-I as it is associated with all pathogenic SPTLC1 and SPTLC2 mutations reported so far, but also warrant for caution in the interpretation of in vitro data.

Missense mutations (K141N and K141E) in the {alpha}-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.

Plants are obliged to defend themselves against a wide range of biotic and abiotic stresses. Complex regulatory signaling networks mount an appropriate defense response depending on the type of stress that is perceived. In response to abiotic stresses such as drought, cold, and salinity, the function of abscisic acid (ABA) is well documented: elevation of plant ABA levels and activation of ABA-responsive signaling result in regulation of stomatal aperture and expression of stress-responsive genes. In response to pathogens, the role of ABA is more obscure and is a research topic that has long been overlooked. This article aims to evaluate and review the reported modes of ABA action on pathogen defense and highlight recent advances in deciphering the complex role of ABA in plant–pathogen interactions. The proposed mechanisms responsible for positive or negative effects of ABA on pathogen defense are discussed, as well as the regulation of ABA signaling and in planta ABA concentrations by beneficial and pathogenic microorganisms. In addition, the fast-growing number of reports that characterize antagonistic and synergistic interactions between abiotic and biotic stress responses point to ABA as an essential component in integrating and fine-tuning abiotic and biotic stress-response signaling networks.

Spinal muscular atrophy (SMA) refers to a group of genetic disorders characterized by degeneration of anterior horn cells of the spinal cord. Initially, the disease was considered purely as an autosomal recessive condition caused by deleterious SMN1 mutations in 5q13. Nowadays a growing number of conditions are designated as non-5q forms of SMA. Materials and methods: A four-generation Bulgarian family with 11 affected members and one sporadic patient afflicted with autosomal dominant proximal SMA underwent detailed neurological examination and testing of muscle strength. Evaluation of serum creatine phosphokinase (CPK) levels and electromyography (EMG) were performed in 6 subjects. The molecular-genetic analysis encompassed genome-wide linkage analysis and whole-exome sequencing. The clinical onset was in childhood, varying between 1 and 6 years (mean 3.17 ± 1.70 years). The affected presented with delayed motor milestones, such as walking, difficulties in getting upright from a squatting position and in climbing stairs, a waddling gait, and slow running. The weakness was limited to the lower limbs and did not show significant progression over time, given that the oldest affected individuals still remained ambulatory into the fifth decade. Symmetric wasting was most prominent in the hip muscles, whereas distal leg muscles seemed atrophic in the older family members. EMG analysis proved anterior horn involvement. Serum creatine phosphokinase (CPK) levels were mostly normal. The genome-wide linkage analysis and whole-exome sequencing found a heterozygous de novo c.320C>T (p. Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2) in the 11 affected from the four-generation family and c.2321A>G (p. Glu774Gly) in the sporadic case. Our study identifies BICD2 gene mutations as a cause of the increasing number non-5q linked SMA and highlights its importance in the motor neuron function in humans.