Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.

ER proteins decipher the tubulin code to regulate organelle distribution.

Organelles move along differentially modified microtubules to establish and maintain their proper distributions and functions1,2. However, how cells interpret these post-translational microtubule modification codes to selectively regulate organelle positioning remains largely unknown. The endoplasmic reticulum (ER) is an interconnected network of diverse morphologies that extends promiscuously throughout the cytoplasm3, forming abundant contacts with other organelles4. Dysregulation of endoplasmic reticulum morphology is tightly linked to neurologic disorders and cancer5,6. Here we demonstrate that three membrane-bound endoplasmic reticulum proteins preferentially interact with different microtubule populations, with CLIMP63 binding centrosome microtubules, kinectin (KTN1) binding perinuclear polyglutamylated microtubules, and p180 binding glutamylated microtubules. Knockout of these proteins or manipulation of microtubule populations and glutamylation status results in marked changes in endoplasmic reticulum positioning, leading to similar redistributions of other organelles. During nutrient starvation, cells modulate CLIMP63 protein levels and p180-microtubule binding to bidirectionally move endoplasmic reticulum and lysosomes for proper autophagic responses.

De novo variants cause complex symptoms in HSP-ATL1 (SPG3A) and uncover genotype-phenotype correlations.

Pathogenic variants in ATL1 are a known cause of autosomal-dominantly inherited hereditary spastic paraplegia (HSP-ATL1, SPG3A) with a predominantly 'pure' HSP phenotype. Although a relatively large number of patients have been reported, no genotype-phenotype correlations have been established for specific ATL1 variants. Confronted with five children carrying de novo ATL1 variants showing early, complex and severe symptoms, we systematically investigated the molecular and phenotypic spectrum of HSP-ATL1. Through a cross-sectional analysis of 537 published and novel cases, we delineate a distinct phenotype observed in patients with de novo variants. Guided by this systematic phenotyping approach and structural modelling of disease-associated variants in atlastin-1, we demonstrate that this distinct phenotypic signature is also prevalent in a subgroup of patients with inherited ATL1 variants and is largely explained by variant localization within a three-dimensional mutational cluster. Establishing genotype-phenotype correlations, we find that symptoms that extend well beyond the typical pure HSP phenotype (i.e. neurodevelopmental abnormalities, upper limb spasticity, bulbar symptoms, peripheral neuropathy and brain imaging abnormalities) are prevalent in patients with variants located within this mutational cluster.

Neuropathy target esterase activity defines phenotypes among PNPLA6 disorders.

Biallelic pathogenic variants in the PNPLA6 gene cause a broad spectrum of disorders leading to gait disturbance, visual impairment, anterior hypopituitarism and hair anomalies. PNPLA6 encodes neuropathy target esterase (NTE), yet the role of NTE dysfunction on affected tissues in the large spectrum of associated disease remains unclear. We present a systematic evidence-based review of a novel cohort of 23 new patients along with 95 reported individuals with PNPLA6 variants that implicate missense variants as a driver of disease pathogenesis. Measuring esterase activity of 46 disease-associated and 20 common variants observed across PNPLA6-associated clinical diagnoses unambiguously reclassified 36 variants as pathogenic and 10 variants as likely pathogenic, establishing a robust functional assay for classifying PNPLA6 variants of unknown significance. Estimating the overall NTE activity of affected individuals revealed a striking inverse relationship between NTE activity and the presence of retinopathy and endocrinopathy. This phenomenon was recaptured in vivo in an allelic mouse series, where a similar NTE threshold for retinopathy exists. Thus, PNPLA6 disorders, previously considered allelic, are a continuous spectrum of pleiotropic phenotypes defined by an NTE genotype:activity:phenotype relationship. This relationship, and the generation of a preclinical animal model, pave the way for therapeutic trials, using NTE as a biomarker.

AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.

Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.

Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A) and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, whereas its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, on the basis of reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.

Ligand-free mitochondria-localized mutant AR-induced cytotoxicity in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

The clinical and molecular spectrum of ZFYVE26-associated hereditary spastic paraplegia: SPG15.

In the field of hereditary spastic paraplegia (HSP), progress in molecular diagnostics needs to be translated into robust phenotyping studies to understand genetic and phenotypic heterogeneity and to support interventional trials. ZFYVE26-associated hereditary spastic paraplegia (HSP-ZFYVE26, SPG15) is a rare, early-onset complex HSP, characterized by progressive spasticity and a variety of other neurological symptoms. While prior reports, often in populations with high rates of consanguinity, have established a general phenotype, there is a lack of systematic investigations and a limited understanding of age-dependent manifestation of symptoms. Here we delineate the clinical, neuroimaging and molecular features of 44 individuals from 36 families, the largest cohort assembled to date. Median age at last follow-up was 23.8 years covering a wide age range (11-61 years). While symptom onset often occurred in early childhood [median: 24 months, interquartile range (IQR) = 24], a molecular diagnosis was reached at a median age of 18.8 years (IQR = 8), indicating significant diagnostic delay. We demonstrate that most patients present with motor and/or speech delay or learning disabilities. Importantly, these developmental symptoms preceded the onset of motor symptoms by several years. Progressive spasticity in the lower extremities, the hallmark feature of HSP-ZFYVE26, typically presents in adolescence and involves the distal lower limbs before progressing proximally. Spasticity in the upper extremities was seen in 64%. We found a high prevalence of extrapyramidal movement disorders including cerebellar ataxia (64%) and dystonia (11%). Parkinsonism (16%) was present in a subset and showed no sustained response to levodopa. Cognitive decline and neurogenic bladder dysfunction progressed over time in most patients. A systematic analysis of brain MRI features revealed a common diagnostic signature consisting of thinning of the anterior corpus callosum, signal changes of the anterior forceps and non-specific cortical and cerebellar atrophy. The molecular spectrum included 45 distinct variants, distributed across the protein structure without mutational hotspots. Spastic Paraplegia Rating Scale scores, SPATAX Disability Scores and the Four Stage Functional Mobility Score showed moderate strength in representing the proportion of variation between disease duration and motor dysfunction. Plasma neurofilament light chain levels were significantly elevated in all patients (Mann-Whitney U-test, P < 0.0001) and were correlated inversely with age (Spearman's rank correlation coefficient r = -0.65, P = 0.01). In summary, our systematic cross-sectional analysis of HSP-ZFYVE26 patients across a wide age-range, delineates core clinical, neuroimaging and molecular features and identifies markers of disease severity. These results raise awareness to this rare disease, facilitate an early diagnosis and create clinical trial readiness.

A class of dynamin-like GTPases involved in the generation of the tubular ER network.

The endoplasmic reticulum (ER) consists of tubules that are shaped by the reticulons and DP1/Yop1p, but how the tubules form an interconnected network is unknown. Here, we show that mammalian atlastins, which are dynamin-like, integral membrane GTPases, interact with the tubule-shaping proteins. The atlastins localize to the tubular ER and are required for proper network formation in vivo and in vitro. Depletion of the atlastins or overexpression of dominant-negative forms inhibits tubule interconnections. The Sey1p GTPase in S. cerevisiae is likely a functional ortholog of the atlastins; it shares the same signature motifs and membrane topology and interacts genetically and physically with the tubule-shaping proteins. Cells simultaneously lacking Sey1p and a tubule-shaping protein have ER morphology defects. These results indicate that formation of the tubular ER network depends on conserved dynamin-like GTPases. Since atlastin-1 mutations cause a common form of hereditary spastic paraplegia, we suggest ER-shaping defects as a neuropathogenic mechanism.

Inhibiting mitochondrial fission rescues degeneration in hereditary spastic paraplegia neurons.

Hereditary spastic paraplegias are characterized by lower limb spasticity resulting from degeneration of long corticospinal axons. SPG11 is one of the most common autosomal recessive hereditary spastic paraplegias, and the SPG11 protein spatacsin forms a complex with the SPG15 protein spastizin and heterotetrameric AP5 adaptor protein complex, which includes the SPG48 protein AP5Z1. Using the integration-free episomal method, we established SPG11 patient-specific induced pluripotent stem cells (iPSCs) from patient fibroblasts. We differentiated SPG11 iPSCs, as well as SPG48 iPSCs previously established, into cortical projection neurons and examined protective effects by targeting mitochondrial dynamics using P110, a peptide that selectively inhibits mitochondrial fission GTPase Drp1. P110 treatment mitigates mitochondrial fragmentation, improves mitochondrial motility, and restores mitochondrial health and ATP levels in SPG11 and SPG48 neurons. Neurofilament aggregations are increased in SPG11 and SPG48 axons, and these are also suppressed by P110. Similarly, P110 mitigates neurofilament disruption in both SPG11 and SPG48 knockdown cortical projection neurons, confirming the contribution of hereditary spastic paraplegia gene deficiency to subsequent neurofilament and mitochondrial defects. Strikingly, neurofilament aggregations in SPG11 and SPG48 deficient neurons double stain with ubiquitin and autophagy related proteins, resembling the pathological hallmark observed in SPG11 autopsy brain sections. To confirm the cause-effect relationship between the SPG11 mutations and disease phenotypes, we knocked-in SPG11 disease mutations to human embryonic stem cells (hESCs) and differentiated these stem cells into cortical projection neurons. Reduced ATP levels and accumulated neurofilament aggregations along axons are observed, and both are mitigated by P110. Furthermore, rescue experiment with expression of wild-type SPG11 in cortical projection neurons derived from both SPG11 patient iPSCs and SPG11 disease mutation knock-in hESCs leads to rescue of mitochondrial dysfunction and neurofilament aggregations in these SPG11 neurons. Finally, in SPG11 and SPG48 long-term cultures, increased release of phosphoNF-H, a biomarker for nerve degeneration, is significantly reduced by inhibiting mitochondrial fission pharmacologically using P110 and genetically using Drp1 shRNA. Taken together, our results demonstrate that impaired mitochondrial dynamics underlie both cytoskeletal disorganization and axonal degeneration in SPG11 and SPG48 neurons, highlighting the importance of targeting these pathologies therapeutically.

Early-Onset and Severe Complex Hereditary Spastic Paraplegia Caused by De Novo Variants in SPAST.

BACKGROUND: Familial hereditary spastic paraplegia (HSP)-SPAST (SPG4) typically presents with a pure HSP phenotype. OBJECTIVE: The aim of this study was to delineate the genotypic and phenotypic spectrum of children with de novo HSP-SPAST. METHODS: This study used a systematic cross-sectional analysis of clinical and molecular features. RESULTS: We report the clinical and molecular spectrum of 40 patients with heterozygous pathogenic de novo variants in SPAST (age range: 2.2-27.7 years). We identified 19 unique variants (16/40 carried the same recurrent variant, p.Arg499His). Symptom onset was in early childhood (median: 11.0 months, interquartile range: 6.0 months) with significant motor and speech delay, followed by progressive ascending spasticity, dystonia, neurogenic bladder dysfunction, gastrointestinal dysmotility, and epilepsy. The mean Spastic Paraplegia Rating Scale score was 32.8 ± 9.7 (standard deviation). CONCLUSIONS: These results confirm that de novo variants in SPAST lead to a severe and complex form of HSP that differs from classic familial pure HSP-SPAST. Clinicians should be aware of this syndrome in the differential diagnosis for cerebral palsy. © 2022 International Parkinson and Movement Disorder Society.

An integrated modelling methodology for estimating global incidence and prevalence of hereditary spastic paraplegia subtypes SPG4, SPG7, SPG11, and SPG15.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are progressively debilitating neurodegenerative disorders that follow heterogenous patterns of Mendelian inheritance. Available epidemiological evidence provides limited incidence and prevalence data, especially at the genetic subtype level, preventing a realistic estimation of the true social burden of the disease. The objectives of this study were to (1) review the literature on epidemiology of HSPs; and (2) develop an epidemiological model of the prevalence of HSP, focusing on four common HSP genetic subtypes at the country and region-level. METHODS: A model was constructed estimating the incidence at birth, survival, and prevalence of four genetic subtypes of HSP based on the most appropriate published literature. The key model parameters were assessed by HSP clinical experts, who provided feedback on the validity of assumptions. A model was then finalized and validated through comparison of outputs against available evidence. The global, regional, and national prevalence and patient pool were calculated per geographic region and per genetic subtype. RESULTS: The HSP global prevalence was estimated to be 3.6 per 100,000 for all HSP forms, whilst the estimated global prevalence per genetic subtype was 0.90 (SPG4), 0.22 (SPG7), 0.34 (SPG11), and 0.13 (SPG15), respectively. This equates to an estimated 3365 (SPG4) and 872 (SPG11) symptomatic patients, respectively, in the USA. CONCLUSIONS: This is the first epidemiological model of HSP prevalence at the genetic subtype-level reported at multiple geographic levels. This study offers additional data to better capture the burden of illness due to mutations in common genes causing HSP, that can inform public health policy and healthcare service planning, especially in regions with higher estimated prevalence of HSP.

Impaired mitochondrial dynamics underlie axonal defects in hereditary spastic paraplegias.

Mechanisms by which long corticospinal axons degenerate in hereditary spastic paraplegia (HSP) are largely unknown. Here, we have generated induced pluripotent stem cells (iPSCs) from patients with two autosomal recessive forms of HSP, SPG15 and SPG48, which are caused by mutations in the ZFYVE26 and AP5Z1 genes encoding proteins in the same complex, the spastizin and AP5Z1 proteins, respectively. In patient iPSC-derived telencephalic glutamatergic and midbrain dopaminergic neurons, neurite number, length and branching are significantly reduced, recapitulating disease-specific phenotypes. We analyzed mitochondrial morphology and noted a significant reduction in both mitochondrial length and their densities within axons of these HSP neurons. Mitochondrial membrane potential was also decreased, confirming functional mitochondrial defects. Notably, mdivi-1, an inhibitor of the mitochondrial fission GTPase DRP1, rescues mitochondrial morphology defects and suppresses the impairment in neurite outgrowth and late-onset apoptosis in HSP neurons. Furthermore, knockdown of these HSP genes causes similar axonal defects, also mitigated by treatment with mdivi-1. Finally, neurite outgrowth defects in SPG15 and SPG48 cortical neurons can be rescued by knocking down DRP1 directly. Thus, abnormal mitochondrial morphology caused by an imbalance of mitochondrial fission and fusion underlies specific axonal defects and serves as a potential therapeutic target for SPG15 and SPG48.

Cellular pathways of hereditary spastic paraplegia.

Human voluntary movement is controlled by the pyramidal motor system, a long CNS pathway comprising corticospinal and lower motor neurons. Hereditary spastic paraplegias (HSPs) are a large, genetically diverse group of inherited neurologic disorders characterized by a length-dependent distal axonopathy of the corticospinal tracts, resulting in lower limb spasticity and weakness. A range of studies are converging on alterations in the shaping of organelles, particularly the endoplasmic reticulum, as well as intracellular membrane trafficking and distribution as primary defects underlying the HSPs, with clear relevance for other long axonopathies affecting peripheral nerves and lower motor neurons.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

Chronic Dengue Virus Panencephalitis in a Patient with Progressive Dementia with Extrapyramidal Features.

OBJECTIVE: To determine the underlying etiology in a patient with progressive dementia with extrapyramidal signs and chronic inflammation referred to the National Institutes of Health Undiagnosed Diseases Program. METHODS: Extensive investigations included metabolic profile, autoantibody panel, infectious etiologies, genetic screening, whole exome sequencing, and the phage-display assay, VirScan, for viral immune responses. An etiological diagnosis was established postmortem. RESULTS: Using VirScan, enrichment of dengue viral antibodies was detected in cerebrospinal fluid as compared to serum. No virus was detected in serum or cerebrospinal fluid, but postmortem analysis confirmed dengue virus in the brain by immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and sequencing. Dengue virus was also detectable by polymerase chain reaction and sequencing from brain biopsy tissue collected 33 months antemortem, confirming a chronic infection despite a robust immune response directed against the virus. Immunoprofiling and whole exome sequencing of the patient did not reveal any immunodeficiency, and sequencing of the virus demonstrated wild-type dengue virus in the central nervous system. INTERPRETATION: Dengue virus is the most common arbovirus worldwide and represents a significant public health concern. Infections with dengue virus are usually self-limiting, and chronic dengue infections have not been previously reported. Our findings suggest that dengue virus infections may persist in the central nervous system causing a panencephalitis and should be considered in patients with progressive dementia with extrapyramidal features in endemic regions or with relevant travel history. Furthermore, this work highlights the utility of comprehensive antibody profiling assays to aid in the diagnosis of encephalitis of unknown etiology. ANN NEUROL 2019;86:695-703.

Reep1 null mice reveal a converging role for hereditary spastic paraplegia proteins in lipid droplet regulation.

Hereditary spastic paraplegias (HSPs; SPG1-76 plus others) are length-dependent disorders affecting long corticospinal axons, and the most common autosomal dominant forms are caused by mutations in genes that encode the spastin (SPG4), atlastin-1 (SPG3A) and REEP1 (SPG31) proteins. These proteins bind one another and shape the tubular endoplasmic reticulum (ER) network throughout cells. They also are involved in lipid droplet formation, enlargement, or both in cells, though mechanisms remain unclear. Here we have identified evidence of partial lipoatrophy in Reep1 null mice in addition to prominent spastic paraparesis. Furthermore, Reep1-/- embryonic fibroblasts and neurons in the cerebral cortex both show lipid droplet abnormalities. The apparent partial lipodystrophy in Reep1 null mice, although less severe, is reminiscent of the lipoatrophy phenotype observed in the most common form of autosomal recessive lipodystrophy, Berardinelli-Seip congenital lipodystrophy. Berardinelli-Seip lipodystrophy is caused by autosomal recessive mutations in the BSCL2 gene that encodes an ER protein, seipin, that is also mutated in the autosomal dominant HSP SPG17 (Silver syndrome). Furthermore, REEP1 co-immunoprecipitates with seipin in cells. This strengthens the link between alterations in ER morphogenesis and lipid abnormalities, with important pathogenic implications for the most common forms of HSP.

Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease.

Adaptor proteins (AP 1-5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and 'fingerprint bodies'. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.