ZFYVE26/SPASTIZIN and SPG11/SPATACSIN mutations in hereditary spastic paraplegia types AR-SPG15 and AR-SPG11 have different effects on autophagy and endocytosis.

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis. Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A1; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.

Hereditary spastic paraplegia type 5: natural history, biomarkers and a randomized controlled trial.

Spastic paraplegia type 5 (SPG5) is a rare subtype of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative disorders defined by progressive neurodegeneration of the corticospinal tract motor neurons. SPG5 is caused by recessive mutations in the gene CYP7B1 encoding oxysterol-7α-hydroxylase. This enzyme is involved in the degradation of cholesterol into primary bile acids. CYP7B1 deficiency has been shown to lead to accumulation of neurotoxic oxysterols. In this multicentre study, we have performed detailed clinical and biochemical analysis in 34 genetically confirmed SPG5 cases from 28 families, studied dose-dependent neurotoxicity of oxysterols in human cortical neurons and performed a randomized placebo-controlled double blind interventional trial targeting oxysterol accumulation in serum of SPG5 patients. Clinically, SPG5 manifested in childhood or adolescence (median 13 years). Gait ataxia was a common feature. SPG5 patients lost the ability to walk independently after a median disease duration of 23 years and became wheelchair dependent after a median 33 years. The overall cross-sectional progression rate of 0.56 points on the Spastic Paraplegia Rating Scale per year was slightly lower than the longitudinal progression rate of 0.80 points per year. Biochemically, marked accumulation of CYP7B1 substrates including 27-hydroxycholesterol was confirmed in serum (n = 19) and cerebrospinal fluid (n = 17) of SPG5 patients. Moreover, 27-hydroxycholesterol levels in serum correlated with disease severity and disease duration. Oxysterols were found to impair metabolic activity and viability of human cortical neurons at concentrations found in SPG5 patients, indicating that elevated levels of oxysterols might be key pathogenic factors in SPG5. We thus performed a randomized placebo-controlled trial (EudraCT 2015-000978-35) with atorvastatin 40 mg/day for 9 weeks in 14 SPG5 patients with 27-hydroxycholesterol levels in serum as the primary outcome measure. Atorvastatin, but not placebo, reduced serum 27-hydroxycholesterol from 853 ng/ml [interquartile range (IQR) 683-1113] to 641 (IQR 507-694) (-31.5%, P = 0.001, Mann-Whitney U-test). Similarly, 25-hydroxycholesterol levels in serum were reduced. In cerebrospinal fluid 27-hydroxycholesterol was reduced by 8.4% but this did not significantly differ from placebo. As expected, no effects were seen on clinical outcome parameters in this short-term trial. In this study, we define the mutational and phenotypic spectrum of SPG5, examine the correlation of disease severity and progression with oxysterol concentrations, and demonstrate in a randomized controlled trial that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in serum of SPG5 patients. We thus demonstrate the first causal treatment strategy in hereditary spastic paraplegia.

Clinical and Paraclinical Indicators of Motor System Impairment in Hereditary Spastic Paraplegia: A Pilot Study.

BACKGROUND: Hereditary spastic paraplegias (HSP) are a composite and genetically heterogeneous group of conditions mainly expressed by the impairment of the central motor system ("pure" forms). The involvement of other components of the central nervous system or of other systems is described in the "complicate" forms. The definition of an investigation protocol capable, by assembling clinical and paraclinical indicators to fully represent the extent of the motor system impairment, would help both the clinical handling of these conditions and contribute to our understanding of their pathogenesis. METHODS: We applied a clinical and paraclinical protocol which included tools exploring motor and non motor functioning, neurophysiology and MRI to a composite cohort of 70 molecularly defined HSP patients aged 3 to 65, to define for each indicator its significance in detailing the presence and the severity of the pathology. RESULTS: Clinically increased deep tendon reflexes and lower limb (LL) weakness are constant findings in all patients. The "complicated" forms are characterized by peripheral motor impairment, cognitive and cerebellar involvement. The Spastic Paraplegia Rating Scale efficiently reflects the severity of functional problems and correlates with disease duration. Neurophysiology consistently documents the impairment of the central motor pathway to the LLs. Nevertheless, the upper extremities and sensory system involvement is a frequent finding. MRI diffusion tensor imaging (DTI) highlighted a significant alteration of FA and MD. Combining the sampling of the various portion of the cortico-spinal tract (CST) DTI consistently discriminated patients from controls. CONCLUSION: We propose a graded clinical and paraclinical protocol for HSP phenotype definition, indicating for each tool the discriminative and descriptive capacity. Our protocol applied to 9 different forms of HSP showed that the functional impairment often extends beyond the CST. The novel DTI approach may add significant elements in disease recognition, staging and mapping.

Variants in KIF1A gene in dominant and sporadic forms of hereditary spastic paraparesis.

KIF1A gene encodes the kinesin 1a protein, an axonal motor protein working in cargo transport along neurites. Variants in KIF1A were identified in different forms of neurodegenerative diseases with dominant and recessive inheritance. Homozygous recessive mutations were found in the hereditary sensory and autonomic neuropathy type 2, HSAN2 and in a recessive subtype of hereditary spastic paraparesis, SPG30. De novo heterozygous dominant variants were found both in a dominant form of SPG30 (AD-SPG30) with one single family reported and in patients with different forms of progressive neurodegenerative diseases. We report the results of a genetic screening of 192 HSP patients, with the identification of four heterozygous variants in KIF1A in four cases, two of whom with family history for the disease. Three of the four variants fall within the motor domain, a frequent target for variants related to the AD-SPG30 subtype. The fourth variant falls downstream the motor domain in a region lacking any functional domain. The KIF1A-related patients show clinical pictures overlapping the known AD-SPG30 phenotype including pure and complicated forms with few differences. Of note, one of the families, originating from the Sicily island, carries the same variant p.S69L detected in the first AD-SPG30 family of Finnish origin reported; differently from the first one, the latter family shows a wide intra-familial phenotype variability. Overall, these data reveal a very low frequency of the AD-SPG30 subtype while confirming the presence of amino acid residues in the motor domain representing preferential targets for mutations, thereby supporting their functional relevance in kinesin 1a activity.

A novel mutation in motor domain of KIF5A associated with an HSP/axonal neuropathy phenotype.

SPG10 is an autosomal dominant hereditary spastic paraplegia (HSP) caused by mutations in the gene KIF5A encoding the heavy chain of kinesin, a motor protein implied in motility functions within cells. Most of the KIF5A mutations are clustered in 2 areas of motor domain of the protein, the switch regions I and II, that are necessary for microtubules interaction. The set of mutations in KIF5A described so far account for a spectrum of clinical heterogeneity ranging from pure HSP to isolated peripheral nerve involvement (Charcot-Marie-Tooth phenotype) or complicated HSP phenotypes. We here describe a patient presenting with progressive walking difficulties and burning dysesthesias, numbness, and pain at distal segments of the 4 limbs. Neurological examination revealed severe spastic gait and vibratory and proprioception sensory reduction in the lower limbs. Motor and sensory nerve conduction studies disclosed axonal damage of peripheral nerves at lower limbs. We identified the novel variant c.967C>T in the KIF5A gene resulting in the R323W change, which is located at the C-terminus of the motor domain of the KIF5A protein, just upstream the linker region but out of the switch regions. Our findings confirm that the "mixed" central-peripheral involvement is the most frequent clinical picture related to KIF5A motor domain mutations and that motor domain "in toto," even outside of the switch regions, is a hot spot for pathogenic mutations. We stress the concept that detection of a peripheral axonal neuropathy in an autosomal dominant HSP patient should be regarded as an important diagnostic tool and should guide clinicians to seek, first of all, KIF5A mutations.

Impairment of brain and muscle energy metabolism detected by magnetic resonance spectroscopy in hereditary spastic paraparesis type 28 patients with DDHD1 mutations.

Mutations in DDHD1 gene have been associated with the SPG28 subtype of Hereditary Spastic Paraparesis (HSP). Clinical phenotype includes axonal neuropathy, distal sensory loss, and cerebellar eye movement disturbances. We screened 96 index subjects from recessive HSP families for mutation and identified one family with two sibs carrying mutations in DDHD1 gene. Clinical, neuropsychological, and neuroimaging studies were performed, including MR spectroscopy of brain and muscle of the two mutated patients. Two novel heterozygous mutations in DDHD1 were found in the affected members of one family, with clinical features overlapping the SPG28 subtype. Of note, MR spectroscopy of brain and muscle in these patients indicated a mild deficit of brain energy metabolism in the oldest and most severely affected patient, while an impairment of energy metabolism was found in the skeletal muscle of both patients. Unlike the DDHD2 mutated patients, no evidence of lipid accumulation in the brain was found. Our data along with those previously reported suggest a dysfunction in the OXPHOS system possibly due to mitochondrial lipid content modification, which could be a central mechanism in the pathogenesis of SPG28.

Hereditary spastic paraparesis in adults. A clinical and genetic perspective from Tuscany.

OBJECTIVE: Hereditary spastic paraparesis or paraplegias (HSPs) are a group of neurogenetic conditions with prominent involvement of the pyramidal tracts. Aim of this study is the clinical and molecular characterization of a cohort of patients with HSP. Moreover, we aim to study the minimum prevalence of HSP in our area and to propose a schematic diagnostic approach to HSP patients based on the available data from the literature. METHODS: Retrospective/perspective study on the subjects with clinical signs and symptoms indicative of pure or complicated HSP, in whom other possible diagnosis were excluded by appropriate neuroradiological, neurophysiologic and laboratory studies, who have been evaluated by the Neurogenetic Service of our Clinic in last two years (2011-2012). RESULTS: 45 patients were identified. The minimum prevalence of HSP in our area was of about 2.17-3.43/100,000. The SF-36 (quality of life) and SPRS (disease progression) scores were inversely related; the time-saving, four-stage scale of motor disability could predict the SPRS scores with a high statistical significance, and we encourage its use in HSP. Our study confirms SPG4 as the major cause of HSP. All SPG4 patients had a pure HSP phenotype, and the dominant inheritance was evident in the great majority of these subjects. SPG7 was the second genetic cause. Other genotypes were rarer (SPG10, SPG11, SPG17). CONCLUSION: Exact molecular diagnosis will allow a more accurate patient counseling and, hopefully, will lead to specific, targeted, therapeutic options for these chronic, still incurable diseases.

Mutations in CYP2U1, DDHD2 and GBA2 genes are rare causes of complicated forms of hereditary spastic paraparesis.

Complicated hereditary spastic paraplegias (HSP) are a heterogeneous group of HSP characterized by spasticity associated with a variable combination of neurologic and extra-neurologic signs and symptoms. Among them, HSP with thin corpus callosum and intellectual disability is a frequent subtype, often inherited as a recessive trait (ARHSP-TCC). Within this heterogeneous subgroup, SPG11 and SPG15 represent the most frequent subtypes. We analyzed the mutation frequency of three genes associated with early-onset forms of ARHSP with and without TCC, CYP2U1/SPG56, DDHD2/SPG54 and GBA2/SPG46, in a large population of selected complicated HSP patients by using a combined approach of traditional-based and amplicon-based high-throughput pooled-sequencing. Three families with mutations were identified, one for each of the genes analyzed. Novel homozygous mutations were identified in CYP2U1 (c.1A>C/p.Met1?) and in GBA2 (c.2048G>C/p.Gly683Arg), while the homozygous mutation found in DDHD2 (c.1978G>C/p.Asp660His) had been previously reported in a compound heterozygous state. The phenotypes associated with the CYP2U1 and DDHD2 mutations overlap the SPG56 and the SPG54 subtypes, respectively, with few differences. By contrast, the GBA2 mutated patients show phenotypes combining typical features of both the SPG46 subtype and the recessive ataxia form, with marked intrafamilial variability thereby expanding the spectrum of clinical entities associated with GBA2 mutations. Overall, each of three genes analyzed shows a low mutation frequency in a general population of complicated HSP (<1 % for either CYP2U1 or DDHD2 and approximately 2 % for GBA2). These findings underline once again the genetic heterogeneity of ARHSP-TCC and the clinical overlap between complicated HSP and the recessive ataxia syndromes.

A clinical, genetic, and biochemical characterization of SPG7 mutations in a large cohort of patients with hereditary spastic paraplegia.

Mutations in the SPG7 gene encoding a mitochondrial protein termed paraplegin, are responsible for a recessive form of hereditary spastic paraparesis. Only few studies have so far been performed in large groups of hereditary spastic paraplegia (HSP) patients to determine the frequency of SPG7 mutations. Here, we report the result of a mutation screening conducted in a large cohort of 135 Italian HSP patients with the identification of six novel point mutations and one large intragenic deletion. Sequence analysis of the deletion breakpoint, together with secondary structure predictions of the deleted region, indicate that a complex rearrangement, likely caused by extensive secondary structure formation mediated by the short interspersed nuclear element (SINE) retrotransposons, is responsible for the deletion event. Biochemical studies performed on fibroblasts from three mutant patients revealed mild and heterogeneous mitochondrial dysfunctions that would exclude a specific association of a complex I defect with the pathology at the fibroblast level. Overall, our data confirm that SPG7 point mutations are rare causes of HSP, in both sporadic and familial forms, while underlying the puzzling and intriguing aspects of histological and biochemical consequences of paraplegin loss.

Eight novel mutations in SPG4 in a large sample of patients with hereditary spastic paraplegia.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases. OBJECTIVE: To search for disease-causing mutations in a large series of Italian patients with HSP. DESIGN: Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13. SETTING: Molecular testing facility in Italy. PATIENTS: Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP. MAIN OUTCOME MEASURES: Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13. RESULTS: We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene. CONCLUSIONS: The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP.

Genetic features of antidepressant induced mania and hypo-mania in bipolar disorder.

RATIONALE AND OBJECTIVES: The present study investigated possible genetic association between some polymorphisms possibly involved in antidepressant response and the occurrence of manic or hypo-manic switches during antidepressant treatment. METHODS: We retrospectively examined 169 individuals with a diagnosis of bipolar disorder (BP) type I ( n=103) and II ( n=66), who presented at least one sudden manic or hypo-manic episode (according to DSM IV criteria) during antidepressant therapy, that occurred within a period of 3 weeks from the beginning of the treatment and without any interposed period of well being ("manic switch"). They were compared with a sex, age, and ethnicity-matched group of 247 subjects, randomly selected from our pool of bipolar subjects, who never showed switches. We then randomly selected from the whole sample ("switched" and "not switched") a sub-sample of patients not under mood stabiliser treatment at the time of the index episode (65 "switched" and 117 "not switched") and compared them with a sex, age and ethnicity matched group of 133 subjects, randomly selected from our pool of major depressed patients, who did not present manic switches. The functional polymorphism in the upstream regulatory region of the serotonin transporter (SERTPR), tryptophan hydroxylase (TPH), G-protein beta 3 subunit (Gbeta3), monoamine oxidase A (MAO-A), catechol- O-methyltransferase (COMT), serotonin receptor 2A (5-HT2A), dopamine receptor D2 (DRD2) and dopamine receptor D4 (DRD4) gene variants were analysed using PCR-based techniques. RESULTS AND CONCLUSIONS: The distribution of the genetic polymorphisms was not significantly different between switched and not-switched patients ( P>0.006-Bonferroni corrected). Moreover, no significant difference was found between switched and not switched sub-samples and the sample of major depressed subjects. Further studies are required to investigate other possible related genetic variants influencing the timing of manic-depressive cycle.