Search

Defective autophagy has been linked to lipotoxicity in several cellular models. We aimed to investigate autophagy in lipid-stimulated hepatoma (Huh7) cells and tested whether 4-phenyl butyric acid (4-PBA), a chemical chaperone, has a beneficial role in hepatic fat accumulation and lipotoxicity. We report that long-term (24 h) exposure of hepatocytes to palmitate block autophagic flux that leads to lipid accumulation and cell death. Western blotting analysis showed increased accumulation of SQSTM1/p62, and decreased expression of Beclin1 and Atg7 in palmitate-treated cells. Autophagy inhibition by 3-methyladenine (3-MA) in palmitate-treated cells neither increased SQSTMI/p62 accumulation nor cell death, thus suggesting complete blockade of autophagy by palmitate. 4-PBA reduced lipid accumulation and cell death that were associated with restoration of autophagy. siRNA-mediated knockdown of Atg7 and presence of autophagy inhibitors, 3-MA and chloroquine, resulted in the decrease in lipid-lowering effect of 4-PBA, suggesting that 4-PBA mediates its lipid-lowering effect via autophagy. Apoptotic parameters, including altered Bcl2:Bax ratio and PARP1 cleavage induced by palmitate, were improved by 4-PBA. Our results indicate that palmitate impairs autophagy and increases lipid accumulation in Huh7 cells, whereas 4-PBA plays a protective role in lipid accumulation and lipotoxicity through activation of autophagy.

Li-Fraumeni syndrome (LFS) is associated with the high risk of a diverse spectrum of childhood and adult-onset malignancies with a predominance of the soft-tissue sarcomas, osteosarcoma, breast can...

Lentil Stemphylium blight is a fungal disease threatening sustainable lentil-based cropping in South Asia including Bangladesh. A weather-based model ʻStempediaʼ was developed to understand the risk of the disease. Daily maximum temperature and sunshine hours are the two most important parameters of the model. When tested with field data from Bangladesh and Nepal, the model simulated the disease severity well both without (R 2 = 0.89, n = 17) and with (R 2 = 0.80, n = 18) fungicide application scenarios. As a result, Stempedia was subjected to experimentation to explore the risk and risk management options related to the disease under Bangladesh conditions. Taking Gazipur - a central regional district - as an example, the model identified that, predominantly, gradual reductions in sunshine hours (or, increased cloudiness) during the early part of the ‘disease susceptible window’ had increased the disease severity over the last 30 years. The model simulated Ishurdi, a north-western regional district, as more disease prone than Gazipur and Jessore, a south-eastern regional district. Stempedia simulated seasonal variability of the disease and predicted reduced severity with delayed sowing, the magnitude of which differed between regions. Stempedia also predicted financial returns with three foliar applications of an effective fungicide but this depended on the timing of application. The best financial return occurred with an early fungicide application, as soon as the disease appeared during initiation of flowering, followed by further application(s) if a disease-producing environment was expected. We conclude that the model has potential to assess regional and seasonal risks of this disease and to determine risk aversion by manipulating sowing time. This model can further be used to formulate the principle of foliar fungicide application to achieve financial gain.

The present study investigates the possible anti-nociceptive effect of intraperitoneal (i.p.) honokiol: a phenolic compound originally isolated from Magnolia officinalis, in acute and chronic inflammatory pain models. Doses of 0.1, 5, and 10 mg/kg honokiol were administered in carrageenan induced pain and the dose (honokiol 10 mg/kg i.p.) with most significant response among behavioral tests was selected for further experiments. The i.p. administration of honokiol inhibits mechanical hyperalgesia, mechanical allodynia, and thermal hyperalgesia, without causing any apparent toxicity. To elucidate the effect of honokiol on various cytokines and antioxidant enzymes, quantitative real-time-PCR was performed to determine the expression levels of pro-inflammatory cytokines and antioxidant enzymes. It is demonstrated that honokiol significantly reduced the expression levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF). Similarly, honokiol was also found to potentiate the expression of nuclear factor erythroid 2–related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), and heme oxygenase-1 (HO-1) levels. Additionally, honokiol significantly reduced plasma nitrite levels as compared to complete Freund’s adjuvant (CFA) induced group. X-ray analysis and hematoxylin and eosin (H&E) staining of inflamed and treated paws showed that honokiol reduced the inflammation with significantly less leukocyte infiltration and soft tissue inflammation. In order to explore the possible mechanism of action of honokiol, agonists [piroxicam (5 mg/kg), tramadol (50 mg/kg), and gabapentin (5 mg/kg) i.p.] as well as antagonists [naloxone (4 mg/kg), olanzapine (10 mg/kg), and flumazenil (0.2 mg/kg) i.p.] were used to study involvement of various receptors on the anti-nociceptive effect of honokiol. The potential side effects of honokiol on muscle activity were assessed. An adverse effect testing of honokiol by liver and renal functions were also carried out. The effect of oral honokiol was also assessed on gastrointestinal (GIT) mucosa. Our results demonstrate that honokiol has a significant anti-nociceptive activity through inhibition of anti-inflammatory mediators.

Background GNE myopathy is an adult onset recessive genetic disorder that affects distal muscles sparing the quadriceps. GNE gene mutations have been identified in GNE myopathy patients all over the world. Homozygosity is a common feature in GNE myopathy patients worldwide. Objectives The major objective of this study was to investigate the mutation spectrum of GNE myopathy in India in relation to the population diversity in the country. Materials and methods We have collated GNE mutation data of Indian GNE myopathy patients from published literature and from recently identified patients. We also used data of people of Indian subcontinent from 1000 genomes database, South Asian Genome database and Strand Life Science database to determine frequency of GNE mutations in the general population. Results A total of 67 GNE myopathy patients were studied, of whom 21% were homozygous for GNE variants, while the rest were compound heterozygous. Thirty-five different mutations in the GNE gene were recorded, of which 5 have not been reported earlier. The most frequent mutation was p.Val727Met (65%) found mainly in the heterozygous form. Another mutation, p.Ile618Thr was also common (16%) but was found mainly in patients from Rajasthan, while p.Val727Met was more widely distributed. The latter was also seen at a high frequency in general population of Indian subcontinent in all the databases. It was also present in Thailand but was absent in general population elsewhere in the world. Conclusion p.Val727Met is likely to be a founder mutation of Indian subcontinent.

The aim of the present investigation was to study the events associated with palmitic acid induced metabolic and lipotoxic changes in human hepatoma cells (Hep3B, Huh7 and HepG2). The cells were lipid-overloaded with varied concentrations of saturated fatty acid (palmitic acid). It was observed that (a) SFA induced significant metabolic changes including CD36/FAT, SREBP1, PPARγ and SCD1 upregulation and decreased Mttp levels in HepG2 and Huh7 cells but not in Hep3B cells; (b) the toxic manifestations including changes in Ca2+ levels, mitochondrial dysfunction and activation of the PERK-eIF2α-CHOP pathway due to SFA were more pronounced in Huh7 and Hep3B cells as compared to HepG2 cells; (c) induction of CYP2E1 and oxidative stress by SFA was observed in all three hepatoma cells. Interestingly, CYP2E1 overexpressing cells (E47 cells) were found to be significantly sensitized towards lipotoxicity; and (d) metabolic and toxic manifestations including altered blood biochemistry, ER stress and CYP2E1 induction were evident in C57BL/6J mice fed with a diet rich in cholesterol and saturated fatty acids (2% cholesterol/12% SFA-HC/HF) for 16 weeks (the model used to confirm the key events taking place in the in vitro system). Histopathological analysis of livers of HC/HF fed mice showed micro- and macro-vesicular steatosis, hepatocyte ballooning, infiltration of neutrophils and prominence of Kupffer cells. In conclusion our data suggest that palmitic acid alters lipid metabolism, causes oxidative stress and disturbs the intracellular Ca2+ balance that terminates in CYP2E1 induction, ER stress and lipoapoptosis.

Inherited retinal degeneration (IRD) are a group of genetically heterogeneous disease of which retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are the most common and severe type. In our study we had taken three unrelated South Indian consanguineous IRD families. Homozygosity mapping was done using Affymetrix 250K Nsp1 GeneChip in each of LCA, Cone-Rod dystrophy (CRD) and autosomal recessive RP (arRP) families followed by targeted re-sequencing by next generation sequencing (NGS) on Illumina MiSeq. Known candidate genes ranging from 1-8 in numbers within the homozygous blocks were identified by homozygosity mapping and targeted NGS revealed the causative mutations; RDH12 c.832A>C, ABCA4 c.1462G>T, CDHR1c.1384_1392delCTCCTGGACinsG, in the LCA, CRD and arRP families, respectively. The identified mutations were validated by Sanger sequencing, segregation in the families and their absence in 200 control chromosomes. Homozygosity mapping guided targeted NGS, especially when more numbers of known candidate genes within the homozygous blocks are observed is a comprehensive method for mutation identification. Molecular data from a larger retinal degenerative disease cohort would reveal the spectrum and prevalence of mutations and genes in Indian population. Molecular diagnosis also aids in genetic counseling, offering carrier and prenatal testing to family members.

The MECP2 gene, located at Xq28, encodes methyl-CpG-binding protein 2 (MeCP2), which is frequently mutated (up to 90%) in Rett syndrome (RTT). RTT is a progressive neurodevelopmental disorder, which affects primarily girls during early childhood and it is one of the most common causes of mental retardation in females. R270X is one of the most frequent recurrent MECP2 mutations among RTT cohorts. The R270X mutation resides within the TRD-NLS (Transcription Repression Domain-Nuclear Localization Signal) region of MeCP2 and causes a more severe clinical phenotype with increased mortality as compared to other mutations. To evaluate the functional role of the R270X mutation, we generated a transgenic mouse model expressing MeCP2270_EGFP (human mutation equivalent) by BAC recombineering. The expression pattern of MeCP2270_EGFP was similar to that of endogenous MeCP2. Strikingly, MeCP2270_EGFP localizes in the nucleus, contrary to the conjecture that R270X could cause disruption of the NLS. In primary hippocampal cells, we show that MeCP2270_EGFP was expressed in astrocytes by colocalization with the astrocyte-specific marker glial fibrillary acidic protein. Our data showing expression of MeCP2270_EGFP in transgenic mice astrocytes further reinforce the recent findings concerning the expression of MeCP2 in the glial cells.

Four homologs to the Drosophila homeotic gene spalt (sal) exist in both humans and mice (SALL1 to SALL4/Sall1 to Sall4, respectively). Mutations in both SALL1 and SALL4 result in the autosomal-dominant developmental disorders Townes-Brocks and Okihiro syndrome, respectively. In contrast, no human diseases have been associated with SALL2 to date, and Sall2-deficient mice have shown no apparent abnormal phenotype. We generated mice deficient in Sall2 and, contrary to previous reports, 11% of our Sall2-deficient mice showed background-specific neural tube defects, suggesting that Sall2 has a role in neurogenesis. To investigate whether Sall4 may compensate for the absence of Sall2, we generated compound Sall2 knockout/Sall4 genetrap mutant mice. In these mutants, the incidence of neural tube defects was significantly increased. Furthermore, we found a similar phenotype in compound Sall1/4 mutant mice, and in vitro studies showed that SALL1, SALL2, and SALL4 all co-localized in the nucleus. We therefore suggest a fundamental and redundant function of the Sall proteins in murine neurulation, with the heterozygous loss of a particular SALL protein also possibly compensated in humans during development.

The SPG4 gene is frequently mutated in autosomal dominant form of hereditary spastic paraplegia (HSP). We report that the compound heterozygous sequence variants S44L, a known polymorphism, and c.1687G>A, a novel mutation in SPG4 cause a severe form of HSP in a patient. The family members carrying solely c.1687G>A mutation are asymptomatic for HSP. The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the c.1687G>A mutation is a splice site mutation and causes skipping of the exon 15 of spastin. Furthermore, quantification of RT-PCR products by sequencing and quantification of allele-specific expression by pyrosequencing assay revealed that c.1687G>A is a leaky or hypomorphic splice site mutation. At the protein level, c.1687G>A mutation in SPG4 leads to E563K substitution. In ex vivo study, about 10% of cells expressing E563K mutant spastin showed filamentous expression pattern, suggesting a hypomorphic effect at the protein level. Collectively, our results suggest that S44L in association with c.1687G>A (E563K) drops the functional level of spastin below a threshold limit sufficient to manifest HSP.

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum (ARHSP-TCC) is one of the most prevalent forms of complex ARHSP. Mutations in the SPG11 gene are the most common cause for ARHSP-TCC and accounts for up to 70% of all cases. The mutational spectrum of SPG11 gene is broad as all types of DNA alterations are detected in the gene and most mutations lead to a premature truncation of the protein, suggesting “loss of function” as the likely pathogenic mechanism. In the current study, we report a consanguineous Turkish family with ARHSP inheritance manifesting white matter abnormalities including TCC with relatively late age of onset. Sequencing of SPG11 gene revealed a homozygous insertion of 15 nucleotides at position 6886 in exon 38 (c.6886_6900Dup15) leading to an in-frame insertion of five amino acids at codon 2296 (p.K2296_L2300Dup5), which resides within a predicted, highly conserved, intradiol ring-cleavage dioxygenase domain (2104 -2381 residues). In silico structural prediction of intradiol domain of the mutated Spatacsin protein revealed that the duplication of five amino acids leads to an extra turn in α-helix and a slightly longer loop region. Our structural analysis suggests that it is unlikely that insertion mutation (c.6886_6900Dup15) causes dysfunctional protein rather the minor conformation changes may elicit a “gain of function”, which may be detrimental to endogenous function of Spatacsin thus cause HSP.

Spastin, the most commonly mutated protein in the autosomal dominant form of hereditary spastic paraplegia (AD-HSP) has been suggested to be involved in vesicular cargo trafficking; however, a comprehensive function of spastin has not yet been elucidated. To characterize the molecular function of spastin, we used the yeast two-hybrid approach to identify new interacting partners of spastin. Here, we report ZFYVE27, a novel member of the FYVE-finger family of proteins, as a specific spastin-binding protein, and we validate the interaction by both in vivo coimmunoprecipitation and colocalization experiments in mammalian cells. More importantly, we report a German family with AD-HSP in which ZFYVE27 (SPG33) is mutated; furthermore, we demonstrate that the mutated ZFYVE27 protein shows an aberrant intracellular pattern in its tubular structure and that its interaction with spastin is severely affected. We postulate that this specific mutation in ZFYVE27 affects neuronal intracellular trafficking in the corticospinal tract, which is consistent with the pathology of HSP.

The murine calgizzarin like gene (Cal) encodes for a calcium binding protein, which belongs to the S100 family of EF-hand proteins. It is specifically expressed in Sertoli cells in the testis and its expression is down-regulated by unknown factor(s) from spermatocytes/spermatids. In this paper, we show by transfection of a fusion protein of green fluorescent protein and Cal protein into NIH3T3 cells, that the expression of Cal is restricted only in the cytoplasm of the cell. A differentially regulated cytoplasmic expression of the Cal in Sertoli cells during mouse development suggests that Cal might play an important role during spermatogenesis. In order to elucidate the function of the Cal protein in the spermatogenesis, we disrupted the Cal locus in mouse by homologous recombination. In our knockout mouse, we deleted exon 2 and exon 3 of the Cal gene and replaced them with a neomycin cassette, which resulted in a complete loss of the Cal transcript. Male and female Cal4+/− and Cal4−/− mice from genetic backgrounds C57BL/6J × 129X1/SvJ hybrid and 129X1/SvJ inbred exhibited normal phenotype and were fertile. An intensive phenotypic analysis showed no gross abnormalities in testis morphology. The lack of the Cal protein also does not affect the parameters of sperm, as they are able to fertilize the oocytes in a competent manner, which is comparable to wild-type sperm. Collectively our results demonstrate that Cal is a nonessential protein and it does not play an important role in mouse spermatogenesis or in process of fertilization. Mol. Reprod. Dev. 66: 431–438, 2003. © 2003 Wiley-Liss, Inc.

The testicular haploid expressed gene (Theg) encodes for a novel ;42.0-kDa nuclear protein, which is specifically expressed in spermatid cells. Its expression is upregulated by some unknown factor(s) from Sertoli cells. To elucidate the function of Theg protein and its role in spermatogenesis, we disrupted the Theg locus in mouse by homologous recombination. For functional dissection of the domain structure of the Theg protein, two different knockout approaches were undertaken. In the first knockout mouse (Th14), the C-terminal region of the Theg protein (amino acids 137‐376) was deleted. Both Th141/2 and Th142/2 mice from genetic backgrounds of C57BL/6J 3 129X1/ SvJ hybrid and 129X1/SvJ inbred exhibited a normal phenotype and were fertile. The testes of Th142/2 mice were smaller than those of Th141/2 and Th141/1 mice; however, the testicular morphology and the properties of sperm, including morphology and motility, from Th142/2 mice were similar to those of Th141/2 and Th141/1 mice. These results demonstrate that the C-terminal region of Theg (amino acids 137‐376) does not play an important role in progression of spermatogenesis. In the second knockout mouse (Th15), we deleted the N-terminal domain of the Theg protein, which resulted in complete loss of Theg transcripts. Both Th151/2 and Th152/2 mice from genetic backgrounds C57BL/6J 3 129X1/SvJ hybrid, C3H/J congenic, and 129X1/SvJ inbred appeared normal and were fertile, with no gross abnormalities detected in testicular morphology or sperm properties. Our results from both knockout mouse model systems clearly illustrate that Theg is not essential for spermatogenesis in the mouse. gamete biology; sertoli cells; sperm; spermatogenesis; testis

An intriguing finding about the gene encoding methyl-CpG binding protein 2 (MeCP2) is that the loss-of-function mutations cause Rett syndrome and duplication (gain-of-function) of MECP2 leads to another neurological disorder termed MECP2 duplication syndrome. To ensure proper neurodevelopment, a precise regulation of MeCP2 expression is critical, and any gain or loss of MeCP2 over a narrow threshold level may lead to postnatal neurological impairment. To evaluate MeCP2 dosage effects, we generated Mecp2(WT_EGFP) transgenic (TG) mouse in which MeCP2 (endogenous plus TG) is mildly overexpressed (approximately 1.5×). The TG MeCP2(WT_EGFP) fusion protein is functionally active, as cross breeding of these mice with Mecp2 knockout mice led to alleviation of major phenotypes in the null mutant mice, including premature lethality. To characterize the Mecp2(WT_EGFP) mouse model, we performed an extensive battery of behavioral tests, which revealed that these mice manifest increased aggressiveness and higher pentylenetetrazole (PTZ)-induced seizure propensity. Evaluation of neuronal parameters revealed a reduction in the number of tertiary branching sites and increased spine density in Mecp2(WT_EGFP) transgenic (TG) neurons. Treatment of TG neurons with epileptogenic compound-PTZ led to a marked increase in amplitude and frequency of calcium spikes. Based on our ex vivo and in vivo data, we conclude that epileptic seizures are manifested as the first symptom when MeCP2 is mildly overexpressed in mice.

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27’s self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27DHR3 failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27DHR3 was co-expressed with wild-type ZFYVE27 (ZFYVE27WT), it exerted a dominant negative effect on ZFYVE27WT as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions. peerReviewed

An autosomal recessive form of hereditary spastic paraplegia (AR-HSP) is primarily caused by mutations in the SPG7 gene, which codes for paraplegin, a subunit of the hetero-oligomeric m-AAA protease in mitochondria. In the current study, sequencing of the SPG7 gene in the genomic DNA of 25 unrelated HSP individuals/families led to the identification of two HSP patients with compound heterozygous mutations (p.G349S/p.W583C and p.A510V/p.N739KfsX741) in the coding sequence of the SPG7 gene. We used a yeast complementation assay to evaluate the functional consequence of novel SPG7 sequence variants detected in the HSP patients. We assessed the proteolytic activity of hetero-oligomeric m-AAA proteases composed of paraplegin variant(s) and proteolytically inactive forms of AFG3L2 (AFG3L2(E575Q) or AFG3L2(K354A)) upon expression in m-AAA protease-deficient yeast cells. We demonstrate that the newly identified paraplegin variants perturb the proteolytic function of hetero-oligomeric m-AAA protease. Moreover, commonly occurring silent polymorphisms such as p.T503A and p.R688Q could be distinguished from mutations (p.G349S, p.W583C, p.A510V, and p.N739KfsX741) in our HSP cohort. The yeast complementation assay thus can serve as a reliable system to distinguish a pathogenic mutation from a silent polymorphism for any novel SPG7 sequence variant, which will facilitate the interpretation of genetic data for SPG7.

The SPAST gene encoding for spastin plays a central role in the genetically heterogeneous group of diseases termed hereditary spastic paraplegia (HSP). In this study, we attempted to expand and refine the genetic and phenotypic characteristics of SPAST associated HSP by examining a large cohort of HSP patients/families. Screening of 200 unrelated HSP cases for mutations in the SPAST gene led to detection of 57 mutations (28.5%), of which 47 were distinct and 29 were novel mutations. The distribution analysis of known SPAST mutations over the structural domains of spastin led to the identification of several regions where the mutations were clustered. Mainly, the clustering was observed in the AAA (ATPases associated with diverse cellular activities) domain; however, significant clustering was also observed in the MIT (microtubule interacting and trafficking), MTBD (microtubule-binding domain) and an N-terminal region (228–269 residues). Furthermore, we used a previously generated structural model of spastin as a framework to classify the missense mutations in the AAA domain from the HSP patients into different structural/functional groups. Our data also suggest a tentative genotype–phenotype correlation and indicate that the missense mutations could cause an earlier onset of the disease.

Spastin, a member of the ATPases associated with various cellular activities (AAA) family of proteins, is the most frequently mutated in hereditary spastic paraplegia. The defining feature of the AAA proteins is a structurally conserved AAA domain which assembles into an oligomer. By chemical cross-linking and gel filtration chromatography, we show that spastin oligomerizes into a hexamer. Furthermore, to gain a comprehensive overview of the oligomeric structure of spastin, we generated a structural model of the AAA domain of spastin using template structure of VPS4B and p97/VCP. The generated model of spastin provided us with a framework to classify the identified missense mutations in the AAA domain from hereditary spastic paraplegia patients into different structural/functional groups. Finally, through co-localization studies in mammalian cells, we show that E442Q mutant spastin acts in a dominant negative fashion and causes redistribution of both wild-type spastin monomer and spastin interacting protein, RTN1 into filamentous microtubule bundles.

Spastin, an ATPase belonging to the AAA family of proteins is most commonly mutated in autosomal dominant hereditary spastic paraplegias (HSP). Spastin is a multifaceted protein with versatile role in cellular events, principally involved in microtubule dynamics. To gain further insight into the molecular function of spastin, we used the yeast two-hybrid approach to identify novel interacting partners of spastin. Using spastin as bait, we identified reticulon 1 (RTN1) and reticulon 3 (RTN3) as potential spastin interacting proteins. RTN1 and RTN3 belong to the reticulon (RTN) gene family, which are primarily expressed in the endoplasmic reticulum. Moreover, RTN1 is known to play a role in vesicular transport processes. Using in vitro and in vivo immunoprecipitation experiments, we were able to demonstrate that RTN1 interacts specifically with spastin. Intracellular distribution studies using immunostaining and overexpression of epitope-tagged protein revealed an obvious colocalization of spastin and RTN1 in discrete vesicles in the cytoplasm. Spastin mediates its interaction with RTN1 through its N-terminal region containing a microtubule-interacting and trafficking domain. It is interesting to note that the aberrant intracellular distribution of a truncated spastin protein was rescued by coexpression with RTN1, which highlights the physiological significance of this interaction. Our findings strengthen the hypothesis that disruption of intracellular vesicular transport processes could cause HSP. It is interesting to note that RTN1 is localized to 14q23.1 where SPG15 locus was mapped. Therefore, we considered RTN1 as a candidate gene for the SPG15 locus, but our mutational analysis possibly excludes RTN1 as causative gene.

We report a novel spontaneous mutation named nax in mice, which exhibit delayed hair appearance and ataxia in a homozygote state. Histological analyses of nax brain revealed an overall impairment of the cerebellar cortex. The classical cortical cytoarchitecture was disrupted, the inner granule cell layer was not obvious, the Purkinje cells were not aligned as a Purkinje cell layer, and Bergmann glias did not span the molecular layer. Furthermore, histological analyses of skin showed that the hair follicles were also abnormal. We mapped the nax locus between marker D2Mit158 and D2Mit100 within a region of 800 kb in the middle of chromosome 2 and identified a missense mutation (Gly244Glu) in Acp2, a lysosomal monoesterase. The Glu244 mutation does not affect the stability of the Acp2 transcript, however it renders the enzyme inactive. Ultrastructural analysis of nax cerebellum showed lysosomal storage bodies in nucleated cells, suggesting progressive degeneration as the underlying mechanism. Identification of Acp2 as the gene mutated in nax mice provides a valuable model system for studying the role of Acp2 in cerebellum and skin homeostasis.

The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18-19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22-23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. peerReviewed

To the Editor: I appreciate this opportunity to respond to the letter by Martignoni et al. In the letter, Martignoni et al. raised certain concerns over our previous finding, where we showed that ZFYVE27 (MIM 610243) interacts with spastin and is mutated in a German family with an autosomal-dominant form of hereditary spastic paraplegia (HSP).1 The index patient of this HSP family was screened for mutations in the SPG4 gene (MIM 604277) and was negative for any mutation.