Biophysical Properties of Novel 1-Deoxy-(Dihydro)ceramides Occurring in Mammalian Cells

Recent advances in the molecular genetics of hereditary peripheral neuropathies have led to changes in the classification of these neuropathies. The hereditary motor and sensory neuropathies (HMSNs) include Charcot-Marie-Tooth disease, both the hypertrophic and neuronal forms of disease among others. The hypertrophic from (HMSN, type 1) is characterized by the presence of abundant omon bulbs pathologically and is caused by mutations in at least four separate genetic loci. The neuronal from (HMSN 2) is characterized by axonal loss on nerve biopsy and is caused by entirely separate genetic loci. The infantile hypertrophic hereditary neuropathy (Déjérine-Sottas disease, HMSN 3), which is characterized by abundant onion bulbs similar to HMSN 1, includes patients with mutations of either PMP-22 or MPZ. Among other hereditary motor and sensory neuropathies, the molecular basis of one form of infantile neuroaxonal dystrophy (Schindler disease) has been identified as absence of α-N-acetyl-D-galactosminidase activity, and the hereditary neuropathy with liability to develop pressure palsies and segmental thickening of myelin layers (tomaculous neuropathy) in volves a deletion of the PMP-2 locus on chromosome 17,. Among the hereditary sensory and autonomic neuropathies (HSANs), only the chromosomal region for the autosomal recessive familial dysautonomia (Riley-Day syndrome, HSAN 3) has been localized (Chromosome 9131-q33). Finally, the gene involved in the Portuguese, Swedish, Jewish, Maryland, Appalachian, German, and Indiana types of familial amyloid neuropathy has been identified as transtbyretin, with many of these separately identified kindreds showing the same mutation and others showing mutations with neuropathy result from mutations in the gelsolin gene and apolipoprotein A-I. The remarkable remarkable recent progress has clarified the molecular basis for the genetic heterogeneity of the clinical syndromes of hereditary peripheral neropathies and clarifies the relationships between genotype and clinical and pathologic phenotype.

First in order to explore the possibilities of building a synthetic artificial cell following a biomimetic approach, we engineered synthetic polymer-based giant unilamellar vesicles (GUVs) with selective membrane permeability. Since the membranes of polymeric GUVs present a high impermeability compared to natural lipid membranes, membranes are selectively permeabilized in a biomimetic approach by the insertion of the small pore-forming peptide gramicidin (gA) as gA biopores are known to allow the transport of protons and monovalent ions. Whilst gA has been inserted in lipid membranes in numerous research studies, the challenge of inserting the bacterial pore into polymer membranes is greater because of the significant difference between the pore length and the thickness of the polymer membrane (more than 3.5 times). Confocal laser scanning microscopy (CLSM) was used to show that neither the size, nor the morphology of the GUVs was affected by successful insertion of gA and further to visualize the pH change inside the cavity of GUVs in real-time by recording videos. In order to demonstrate the successful insertion of gA, a pH-sensitive dye is encapsulated inside the cavity of GUVs and proton gradients between the environment of GUVs and their inner cavity serves to assess the exchange of protons across the membrane upon gA insertion. The results showed that gA was successfully inserted and remained functional in polymer membranes with thickness of 9.2–12.1 nm. Larger membrane thicknesses did not allow gA insertion, and 12.1 nm represents a limit for the mismatch between the pore length and the membrane thickness. Our gA-GUVs are therefore pH-regulating and maintain their integrity in different pH conditions in a cell-like manner. This bio-mimetic approach to use ion channels with specific selectivity for insertion in polymer membranes is an elegant strategy to develop mimics of biomembranes or for supporting the design of bioreactors. 
\nNext, a functional cell mimetic compartment is developed by the insertion of the bacterial membrane protein (OmpF) in thick synthetic polymer membranes of an artificial GUV compartment that encloses the oxidative enzyme horseradish peroxidase. In this manner a simple and robust cell mimic is designed, that supports a rudimental form of metabolism. The biopore serves as a gate, which allows substrates to enter the cavities of the GUVs, where they are converted into the resorufin-like products by the encapsulated enzyme, and then released in the environments of GUVs.
\nOur bio-equipped GUVs facilitate the control of specific catalytic reactions in confined micro-scale spaces mimicking cell size and architecture and thus provide a straightforward approach serving to obtain deeper insights in the real-time of biological processes inside cells. 
\nThis elegant strategy of equipping both GUV membranes and GUV cavities with biomolecules, opens the way towards cell-like compartments as novel materials with bio-functionality is the combination of synthetic micrometer-sized giant unilamellar vesicles (GUVs) with biomolecules because it enables studying the behavior of biomolecules and processes within confined cavities.
\nFinally a visionary strategy for creating the first bioinspired molecular factory with functionality as a real cell-mimic based on micrometer-sized giant plasma membrane vesicles (GPMVs) is addressed. GPMVs are cell-derived giant vesicles consisting of an outer compartment architecture (membrane) and an inner composition, which both directly mirror the composition of cells from which they originate except the larger organelles like for example nuclei and Golgi apparatus making measurements easier and are the closest cell-mimic available on the market up to now. In a step towards the development of bioinspired molecular factories with functionality as cell mimics, we generate the next generation of cell mimics by the production of sophisticated hybrid molecular factories based on GPMVs, which are equipped with a synthetic molecular machinery inside their cavities that provides functionality. Such a hierarchical approach in compartmentalization allows the lower-level synthetic functional compartments encapsulated within the cavity of the GPMV to act as independent anatomically discreet units that specialize in their own function, making them nanoscale versions of nature own organelles.
\nTowards the first bioinspired molecular factory enzyme-equipped polymersomes with a reconstituted membrane protein (OmpF) are encapsulated inside the GPMVs as enzymatic nanocompartment spaces, where they retain their structure and functionality. When substrates were added to the outer solution of the GPMVs, it was shown that they could penetrate both the membrane of the GPMVs and the inner compartment membranes of the synthetic nanoreactors equipped with OmpF pores. In this respect the equipment of the catalytic nanocompartment spaces with OmpF was essential as it allowed the enzyme to perform in the inner cavities. Successful substrate conversion was visualized by following the fluorescent product of the enzymatic reaction (resorufin-like product), which could leave the polymersome and diffuse inside the GPMV cavity. Finally we demonstrate that equipped GPMVs can act as artificial cell mimics – retaining their membrane and inner composition if they are injected into multicellular organisms – Zebrafish embryos. To the best of our knowledge, this is the first time that a molecular factory functioning as a cell-like mimic has been be constructed by using a top down-bottom up approach and has been tested in vivo by taking advantage of the fundamental nature of GPMVs. 
\n

The hereditary sensory and autonomic neuropathies (HSAN) encompass a number of inherited disorders that are associated with sensory dysfunction (depressed reflexes, altered pain and temperature perception) and varying degrees of autonomic dysfunction (gastroesophageal reflux, postural hypotention, excessive sweating). Subsequent to the numerical classification of four distinct forms of HSAN that was proposed by Dyck and Ohta, additional entities continue to be described, so that identification and classification are ongoing. As a group, the HSAN are rare diseases that affect both sexes. HSAN III is almost exclusive to individuals of Eastern European Jewish extraction, with incidence of 1 per 3600 live births. Several hundred cases with HSAN IV have been reported. The worldwide prevalence of HSAN type II is very low. This review focuses on the description of three of the disorders, HSAN II through IV, that are characterized by autosomal recessive inheritance and onset at birth. These three forms of HSAN have been the most intensively studied, especially familial dysautonomia (Riley-Day syndrome or HSAN III), which is often used as a prototype for comparison to the other HSAN. Each HSAN disorder is likely caused by different genetic errors that affect specific aspects of small fiber neurodevelopment, which result in variable phenotypic expression. As genetic tests are routinely used for diagnostic confirmation of HSAN III only, other means of differentiating between the disorders is necessary. Diagnosis is based on the clinical features, the degree of both sensory and autonomic dysfunction, and biochemical evaluations, with pathologic examinations serving to further confirm differences. Treatments for all these disorders are supportive.

Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

We read with interest the description of a family with sensory inheritance. A form of HSAN characterized by late onset sensory ataxia was reported in another Dutch family (Staal ataxic neuropathy of autosomal dominant inheritance (van Dijk et al., 1995). Unfortunately, the Dutch authors omitted and Mechelse, 1978) which, however, showed a recessive mode of inheritance, whereas Robinson et al. (1977) described to discuss our report of a family with similar features which appeared in a Dutch journal (Marbini et al., 1994), classified an autosomal dominant form of HSAN with ataxia, but with onset usually in the first two decades. Additional features as hereditary sensory and autonomic neuropathy (HSAN) with ataxia and late onset. were represented by deafness in our family, and oculomotor disorder in the Dutch family. This probably reflects a The proband was a 66-year-old man who complained of unsteady gait, especially during walking in the dark, by age multisystem involvement, as other patients with HSAN and ataxia showed deafness (Robinson et al., 1977) or oculomotor 60 years. Neurological examination showed severe hearing loss, normal limb muscle bulk and strength, absent deep dysfunction (Staal and Mechelse, 1978). Sural nerve biopsy findings were similar in our case and in the Dutch patient, tendon reflexes in the ankles and marked sensory loss in the extremities, especially for the position sense, with severe showing severe loss of myelinated fibres, and moderate changes of the unmyelinated fibres. The most remarkable sensory ataxia. Investigations, including CT and MRI brain scans, were unremarkable except for sensorineural hearing difference was in the somatosensory evoked potentials, which were severely affected in three members of the Dutch family, loss and abnormal autonomic function tests. Neurophysiological study showed normal EMG, extremely reduced or but normal in our proband. This would point to involvement of dorsal root ganglia sensory neurons and distal involvement absent sensory action potentials (SAP), but only minor alterations in the motor nerves. Median and peroneal somatoin the peripheral nerves, respectively. It is also possible, however, that since the disease focuses on the peripheral sensory evoked potentials were normal. Sural nerve biopsy showed a severe loss of myelinated fibres of all calibres sensory neurons, as suggested by Dyck (1993), degeneration involves either the peripherally and centrally directed axons (622/mm2), whereas the unmyelinated fibre population was relatively less involved (7772/mm2; control 32000/mm2). of the dorsal root ganglion cells, or just the peripheral axons (Thomas, 1982). Biopsy of the peroneus brevis muscle showed normal findings. The patient’s brother, aged 62 years, who also complained In our opinion, these cases may represent a subset of autosomal dominant HSAN, or HSAN 1 described by Dyck of swaying for the last few years, showed marked sensory ataxia, absent Achilles tendon reflexes, and deficit in all (1993). This form is usually characterized by acrodystrophic symptoms with onset in the 2nd–3rd decade, but there is sensation modalities in the lower limbs, with normal EMG and electroneurography of the motor nerves, but extremely great variability in the onset and progression of the disease (Dyck, 1993). Our morphometric findings of profound loss of decreased amplitude or absence of the SAPs. Loss of balance with onset in the sixth decade was also myelinated fibres with a relative preservation of unmyelinated fibres do not fit with previous data on HSAN I by Dyck (loss reported in the father and in another brother, both deceased. Neurological examination was normal in the proband’s sons, of unmyelinated fibres and small myelinated fibres) and by Danon and Carpenter (1985) (uniform loss for every fibre aged 18–40 years, as were clinical and electrophysiological study and autonomic function tests in the 35-year-old type), but rather resemble the pathological features of HSAN II (Nukada et al., 1982). A minor involvement of the daughter of the patient’s 62-year-old brother. The paper of van Dijk et al. (1995), and ours, clearly unmyelinated nociceptive fibres could explain the absence of acrodystrophy, and this could result, in turn, in a delayed define a form of HSAN characterized clinically by sensory ataxia and late onset in the fifth or sixth decade, documented manifestation of the disease. Whether the differences between dominant HSN with ataxia and the classical form of HSAN in two generations, consistent with autosomal dominant

Hereditary sensory and autonomic neuropathies (HSANs) are a group of heterogeneous genetic disorders presenting predominantly with sensory and autonomic dysfunction. They are a diverse group of diseases of the peripheral nervous system characterized by profound distal sensory loss and various autonomic and motor disturbances. The primary objective of this study was to describe the clinical presentation of children with HSAN to paediatricians. We present clinical features and genetic etiology of patients with HSAN followed in a Canadian tertiary paediatric centre, including suggestions for their monitoring, management, and long-term follow-up. A retrospective chart review of all patients with HSAN followed from the years 2000 through 2021 was performed. Collected data consisted of patients' demographics, clinical characteristics, imaging, and management. Eight patients were included. The average age at diagnosis was 3.19 ± 2.83 years. Insensitivity to pain (100%), dysautonomia (100%), global development delay (87.5%), emesis (62.5%), and self-injury (62.5%) were the most prevalent manifestations of HSAN. The most common co-morbidities were gastroesophageal reflux disease (50%), obstructive sleep apnea (37.5%), attention-deficit hyperactivity disorder (37.5%), and iron deficiency (37.5%). Management was multi-disciplinary, involving neurologists, orthopeds, developmental paediatricians, sleep specialists, and psychiatrists. HSANs are a diverse group of diseases, characterized by profound distal sensory loss, acral mutilations, and variable autonomic disturbances. It is important to recognize the diagnosis in the paediatrician's office in order to set up surveillance and prevent complications.

Endosomal DNA release studies using giant unilamellar vesicles as model endosomal membranes

The hereditary neuropathies are classified as in hereditary motor sensory neuropathies (HMSN) and hereditary sensory autonomic neuropathies (HSAN) based on the clinical symptoms.

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders characterized by axonal atrophy and degeneration, exclusively or predominantly affecting the sensory and autonomic neurons. So far, disease-associated mutations have been identified in seven genes: two genes for autosomal dominant (SPTLC1 and RAB7) and five genes for autosomal recessive forms of HSAN (WNK1/HSN2, NTRK1, NGFB, CCT5 and IKBKAP). We performed a systematic mutation screening of the coding sequences of six of these genes on a cohort of 100 familial and isolated patients diagnosed with HSAN. In addition, we screened the functional candidate gene NGFR (p75/NTR) encoding the nerve growth factor receptor. We identified disease-causing mutations in SPTLC1, RAB7, WNK1/HSN2 and NTRK1 in 19 patients, of which three mutations have not previously been reported. The phenotypes associated with mutations in NTRK1 and WNK1/HSN2 typically consisted of congenital insensitivity to pain and anhidrosis, and early-onset ulcero-mutilating sensory neuropathy, respectively. RAB7 mutations were only found in patients with a Charcot-Marie-Tooth type 2B (CMT2B) phenotype, an axonal sensory-motor neuropathy with pronounced ulcero-mutilations. In SPTLC1, we detected a novel mutation (S331F) corresponding to a previously unknown severe and early-onset HSAN phenotype. No mutations were found in NGFB, CCT5 and NGFR. Overall disease-associated mutations were found in 19% of the studied patient group, suggesting that additional genes are associated with HSAN. Our genotype–phenotype correlation study broadens the spectrum of HSAN and provides additional insights for molecular and clinical diagnosis.

To determine the number of employed people in a group of patients with neuromuscular diseases and in 3 separate subgroups (facioscapulo-humeral dystrophy, hereditary motor and sensory neuropathy, and myotonic dystrophy) to investigate any differences in employment status between the patient groups, and to identify factors related to employment status. Cross-sectional study. A total of 591 patients with neuromuscular diseases participated in the study, 138 with facioscapulo-humeral dystrophy, 135 with hereditary motor and sensory neuropathy, and 318 with myotonic dystrophy. Self-report questionnaires, the Checklist Individual Strength (CIS) and the Short Form-36 (SF-36). Of the patients with neuromuscular diseases in the study, 56.7% were employed. Younger age, being male, and higher education contributed significantly to employment status of the neuromuscular diseases group and the hereditary motor and sensory neuropathy and myotonic dystrophy subgroups. Significant between-group differences for employed vs not employed subjects were present in the total neuromuscular diseases group on all subscales of the CIS and SF-36. Factors related to employment status differed for the 3 neuromuscular diseases subgroups. More than half of the patients with neuromuscular diseases were employed. Patients with facioscapulo-humeral dystrophy and patients with hereditary motor and sensory neuropathy were more often employed than patients with myotonic dystrophy. Between-group analyses for differences in baseline factors revealed 11 significant factors related to employment. Multivariate logistic analyses revealed 6 factors contributing to employment for the group of patients with neuromuscular diseases.

Hand strength and fatigue in patients with hereditary motor and sensory neuropathy (types I and II)

We describe five families from different regions in Norway with a late-onset autosomal-dominant hereditary polyneuropathy sharing a heterozygous variant in the SLC12A6 gene. Mutations in the same gene have previously been described in infants with autosomal-recessive hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation (Andermann syndrome), and in a few case reports describing dominantly acting de novo mutations, most of them with onset in childhood. The phenotypes in our families demonstrated heterogeneity. Some of our patients only had subtle to moderate symptoms and some individuals even no complaints. None had CNS manifestations. Clinical and neurophysiological evaluations revealed a predominant sensory axonal polyneuropathy with slight to moderate motor components.In all 10 patients the identical SLC12A6 missense variant, NM_001365088.1 c.1655G>A p.(Gly552Asp), was identified. For functional characterization, the mutant potassium chloride cotransporter 3 was modelled in Xenopus oocytes. This revealed a significant reduction in potassium influx for the p.(Gly552Asp) substitution.Our findings further expand the spectrum of SLC12A6 disease, from biallelic hereditary motor and sensory neuropathy with corpus callosum agenesis and mental retardation and monoallelic early-onset hereditary motor and sensory neuropathy caused by de novo mutations, to late-onset autosomal-dominant axonal neuropathy with predominant sensory deficits.

The hereditary sensory and autonomic neuropathies encompass a number of inherited disorders that are associated with sensory dysfunction and varying degree of autonomic dysfunction. Hereditary sensory and autonomic neuropathy (HSAN) with anhidrosis type IV is the second most common form of hereditary sensory neuropathy. We report here the youngest case diagnosed with HSAN type IV having all features of congenital insensitivity to pain and anhidrosis.

Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.

Hereditary sensory and autonomic neuropathies (HSAN) are rare diseases. Five different types are described. Hereditary sensory and autonomic neuropathy type V, also known as congenital insensitivity to pain, is a rare autosomal recessive disease seen in early childhood. Self-mutilation is an invariable feature of this disorder involving the teeth, tongue, lips, and fingers. This report described the case of a 2-year-old baby boy who had self-mutilating injuries to her tongue and hands, caused by biting. Protective splints were given to the patient to prevent further self-mutilation.