MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

Prevention of experimental myasthenia gravis by nasal administration of synthetic acetylcholine receptor T epitope sequences.

T cell tolerization prevents and improves T cell-mediated experimental autoimmune diseases. We investigated here whether similar approaches could be used for antibody (Ab)-mediated autoimmune diseases. Myasthenia gravis, caused by IgG Ab against muscle acetylcholine receptor (AChR), is perhaps the best characterized of them. We used an animal model, experimental myasthenia gravis induced in C57Bl/6 mice by immunization with Torpedo acetylcholine receptor (TAChR), to demonstrate that nasal administration of synthetic sequences of the TAChR alpha-subunit- forming epitopes recognized by anti-TAChR CD4+ T helper cells (residues alpha150-169, alpha181-200, and alpha360-378), given before and during immunization with TAChR, causes decreased CD4+ responsiveness to those epitopes and to TAChR, reduced synthesis of anti-TAChR Ab, and prevented experimental myasthenia gravis. These effects were not induced by nasal administration of synthetic epitopes of diphtheria toxin. Secretion of IL-2, IL-4, and IL-10 by spleen T cells from TAChR immunized mice, in response to challenge with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to TAChR, while peptide-treated mice had also an AChR-specific Th2 response. The TAChR peptide treatment induced also in vitro anergy to the TAChR of the spleen T cells, which was reversed by IL-2.

Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above.

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells.

OBJECTIVES: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. METHODS: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. RESULTS: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CONCLUSIONS: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.

Interleukin-4 deficiency facilitates development of experimental myasthenia gravis and precludes its prevention by nasal administration of CD4+ epitope sequences of the acetylcholine receptor.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.

Subcutaneous administration of T-epitope sequences of the acetylcholine receptor prevents experimental myasthenia gravis.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.

Simple, inexpensive computerized rodent activity meters.

We describe two approaches for using obsolescent computers, either an IBM PC XT or an Apple Macintosh Plus, to accurately quantify spontaneous rodent activity, as revealed by continuous monitoring of the spontaneous usage of running activity wheels. Because such computers can commonly be obtained at little or no expense, and other commonly available materials and inexpensive parts can be used, these meters can be built quite economically. Construction of these meters requires no specialized electronics expertise, and their software requirements are simple. The computer interfaces are potentially of general interest, as they could also be used for monitoring a variety of events in a research setting.

Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4+ cells.

Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.

Mechanisms by which the I-ABM12 mutation influences susceptibility to experimental myasthenia gravis: a study in homozygous and heterozygous mice.

The I-Abm12 mutation in C57B1/6 (B6) mice yields the B6.C-H-2bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4+ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR alpha subunit which dominate the CD4+ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12 x B6)F1 mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4+ cells recognized the same TAChR alpha subunit peptide sequences (T alpha 150-169, T alpha 181-200 and T alpha 360-378). CD4+ cells from TAChR-sensitized F1 mice were challenged with TAChR and alpha subunit epitope peptides, using F1, B6 or bm12 APC. B6 and F1 APC presented all these Ag efficiently, while bm12 APC presented TAChR and peptide T alpha 150-169 poorly and erratically. Anti-TAChR and anti-alpha subunit epitope CD4+ lines propagated from F1 and B6 mice had similar TcR V beta usage. All lines but those specific for the sequence T alpha 150-169 had unrestricted V beta usage. Anti-T alpha 150-169 lines from both B6 and F1 mice had a strong preferential usage of V beta 6. Anti-T alpha 150-169 lines from F1 mice had also a slightly higher V beta 14 usage. B6, bm12 and F1 mice developed similar anti-TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti-AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4+ cells sensitized to epitopes within the sequence T alpha 150-160, consequent to the inability of the I-Abm12 molecule to present this sequence.

Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor.

The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment.

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice.

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.

Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes.

T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide T alpha 360-378 was co-injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides T alpha 360-378 and T alpha 63-80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide T alpha 360-378. Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.

Residues within the alpha subunit sequence 304-322 of muscle acetylcholine receptor forming autoimmune CD4+ epitopes in BALB/c mice.

BALB/c mice develop myasthenic symptoms after immunization with rodent acetylcholine receptor (AChR). After immunization with Torpedo AChR (TAChR), their CD4+ cells become strongly sensitized against a conserved region of the TAChR alpha subunit sequence (residues alpha 304-322), and cross-react vigorously with the homologous sequences of mouse and human AChR, which are almost identical. Therefore AChR-specific potentially autoreactive CD4+ cells exist in this strain. We immunized BALB/c mice with the synthetic TAChR sequence alpha 304-322. The CD4+ cells thus sensitized responded to TAChR, indicating that they recognize an epitope(s) produced upon TAChR processing. They recognized peptide alpha 304-322 in association with the I-Ad molecule. Anti-alpha 304-322 CD4+ cells cross-reacted well with the corresponding murine and human synthetic sequences. To identify residues involved in formation of an autoimmune epitope(s), CD4+ cells from mice immunized with peptide alpha 304-322 were challenged in vitro with single residue glycine-substituted analogues of this sequence. Substitution of residue W311, and of any residue within the sequence alpha 313-319 (RKVFIDT), consistently and, in some cases, strongly affected the CD4+ cells response. Substitution of residues in the region alpha 311-319 had variable effects in different experiments, and in general affected moderately the CD4+ response. These results suggest that anti-alpha 304-322 CD4+ cells comprise several clones, recognizing overlapping epitopes which share residues alpha 311-319. The importance of the sequence region alpha 311-319 for formation of CD4+ cell epitope(s) was verified by testing CD4+ cells sensitized to T alpha 304-322 with analogues of this sequence, carrying non-conservative substitutions at positions Q310, K314 and D318. Substitution of Q310 had minimal or no effects, while those of K314 or D318 strongly affected the CD4+ cell response.

Polymorphism at the HLA-DQ locus determines susceptibility to experimental autoimmune myasthenia gravis.

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-A beta0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-A beta0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.

Transgenic expression of IL-10 in T cells facilitates development of experimental myasthenia gravis.

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.