NPTX1 mutations trigger endoplasmic reticulum stress and cause autosomal dominant cerebellar ataxia.

With more than 40 causative genes identified so far, autosomal dominant cerebellar ataxias exhibit a remarkable genetic heterogeneity. Yet, half the patients are lacking a molecular diagnosis. In a large family with nine sampled affected members, we performed exome sequencing combined with whole-genome linkage analysis. We identified a missense variant in NPTX1, NM_002522.3:c.1165G>A: p.G389R, segregating with the phenotype. Further investigations with whole-exome sequencing and an amplicon-based panel identified four additional unrelated families segregating the same variant, for whom a common founder effect could be excluded. A second missense variant, NM_002522.3:c.980A>G: p.E327G, was identified in a fifth familial case. The NPTX1-associated phenotype consists of a late-onset, slowly progressive, cerebellar ataxia, with downbeat nystagmus, cognitive impairment reminiscent of cerebellar cognitive affective syndrome, myoclonic tremor and mild cerebellar vermian atrophy on brain imaging. NPTX1 encodes the neuronal pentraxin 1, a secreted protein with various cellular and synaptic functions. Both variants affect conserved amino acid residues and are extremely rare or absent from public databases. In COS7 cells, overexpression of both neuronal pentraxin 1 variants altered endoplasmic reticulum morphology and induced ATF6-mediated endoplasmic reticulum stress, associated with cytotoxicity. In addition, the p.E327G variant abolished neuronal pentraxin 1 secretion, as well as its capacity to form a high molecular weight complex with the wild-type protein. Co-immunoprecipitation experiments coupled with mass spectrometry analysis demonstrated abnormal interactions of this variant with the cytoskeleton. In agreement with these observations, in silico modelling of the neuronal pentraxin 1 complex evidenced a destabilizing effect for the p.E327G substitution, located at the interface between monomers. On the contrary, the p.G389 residue, located at the protein surface, had no predictable effect on the complex stability. Our results establish NPTX1 as a new causative gene in autosomal dominant cerebellar ataxias. We suggest that variants in NPTX1 can lead to cerebellar ataxia due to endoplasmic reticulum stress, mediated by ATF6, and associated to a destabilization of NP1 polymers in a dominant-negative manner for one of the variants.

Implication of folate deficiency in CYP2U1 loss of function.

Hereditary spastic paraplegias are heterogeneous neurodegenerative disorders. Understanding of their pathogenic mechanisms remains sparse, and therapeutic options are lacking. We characterized a mouse model lacking the Cyp2u1 gene, loss of which is known to be involved in a complex form of these diseases in humans. We showed that this model partially recapitulated the clinical and biochemical phenotypes of patients. Using electron microscopy, lipidomic, and proteomic studies, we identified vitamin B2 as a substrate of the CYP2U1 enzyme, as well as coenzyme Q, neopterin, and IFN-α levels as putative biomarkers in mice and fluids obtained from the largest series of CYP2U1-mutated patients reported so far. We also confirmed brain calcifications as a potential biomarker in patients. Our results suggest that CYP2U1 deficiency disrupts mitochondrial function and impacts proper neurodevelopment, which could be prevented by folate supplementation in our mouse model, followed by a neurodegenerative process altering multiple neuronal and extraneuronal tissues.

CYP2U1 activity is altered by missense mutations in hereditary spastic paraplegia 56.

Hereditary spastic paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying three novel CYP2U1 missense variants and the development of an in vitro biochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic, and structural (from a 3D model) characteristics of the over expressed wild-type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings.

Motor neuron degeneration in spastic paraplegia 11 mimics amyotrophic lateral sclerosis lesions.

The most common form of autosomal recessive hereditary spastic paraplegia is caused by mutations in the SPG11/KIAA1840 gene on chromosome 15q. The nature of the vast majority of SPG11 mutations found to date suggests a loss-of-function mechanism of the encoded protein, spatacsin. The SPG11 phenotype is, in most cases, characterized by a progressive spasticity with neuropathy, cognitive impairment and a thin corpus callosum on brain MRI. Full neuropathological characterization has not been reported to date despite the description of >100 SPG11 mutations. We describe here the clinical and pathological features observed in two unrelated females, members of genetically ascertained SPG11 families originating from Belgium and Italy, respectively. We confirm the presence of lesions of motor tracts in medulla oblongata and spinal cord associated with other lesions of the central nervous system. Interestingly, we report for the first time pathological hallmarks of SPG11 in neurons that include intracytoplasmic granular lysosome-like structures mainly in supratentorial areas, and others in subtentorial areas that are partially reminiscent of those observed in amyotrophic lateral sclerosis, such as ubiquitin and p62 aggregates, except that they are never labelled with anti-TDP-43 or anti-cystatin C. The neuropathological overlap with amyotrophic lateral sclerosis, associated with some shared clinical manifestations, opens up new fields of investigation in the physiopathological continuum of motor neuron degeneration.

Loss of association of REEP2 with membranes leads to hereditary spastic paraplegia.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous neurological conditions. Their main pathogenic mechanisms are thought to involve alterations in endomembrane trafficking, mitochondrial function, and lipid metabolism. With a combination of whole-genome mapping and exome sequencing, we identified three mutations in REEP2 in two families with HSP: a missense variant (c.107T>A [p.Val36Glu]) that segregated in the heterozygous state in a family with autosomal-dominant inheritance and a missense change (c.215T>A [p.Phe72Tyr]) that segregated in trans with a splice site mutation (c.105+3G>T) in a family with autosomal-recessive transmission. REEP2 belongs to a family of proteins that shape the endoplasmic reticulum, an organelle that was altered in fibroblasts from an affected subject. In vitro, the p.Val36Glu variant in the autosomal-dominant family had a dominant-negative effect; it inhibited the normal binding of wild-type REEP2 to membranes. The missense substitution p.Phe72Tyr, in the recessive family, decreased the affinity of the mutant protein for membranes that, together with the splice site mutation, is expected to cause complete loss of REEP2 function. Our findings illustrate how dominant and recessive inheritance can be explained by the effects and nature of mutations in the same gene. They have also important implications for genetic diagnosis and counseling in clinical practice because of the association of various modes of inheritance to this new clinico-genetic entity.

Interferon ß induces clearance of mutant ataxin 7 and improves locomotion in SCA7 knock-in mice.

We showed previously, in a cell model of spinocerebellar ataxia 7, that interferon beta induces the expression of PML protein and the formation of PML protein nuclear bodies that degrade mutant ataxin 7, suggesting that the cytokine, used to treat multiple sclerosis, might have therapeutic value in spinocerebellar ataxia 7. We now show that interferon beta also induces PML-dependent clearance of ataxin 7 in a preclinical model, SCA7(266Q/5Q) knock-in mice, and improves motor function. Interestingly, the presence of mutant ataxin 7 in the mice induces itself the expression of endogenous interferon beta and its receptor. Immunohistological studies in brains from two patients with spinocerebellar ataxia 7 confirmed that these modifications are also caused by the disease in humans. Interferon beta, administered intraperitoneally three times a week in the knock-in mice, was internalized with its receptor in Purkinje and other cells and translocated to the nucleus. The treatment induced PML protein expression and the formation of PML protein nuclear bodies and decreased mutant ataxin 7 in neuronal intranuclear inclusions, the hallmark of the disease. No reactive gliosis or other signs of toxicity were observed in the brain or internal organs. The performance of the SCA7(266Q/5Q) knock-in mice was significantly improved on two behavioural tests sensitive to cerebellar function: the Locotronic® Test of locomotor function and the Beam Walking Test of balance, motor coordination and fine movements, which are affected in patients with spinocerebellar ataxia 7. In addition to motor dysfunction, SCA7(266Q/5Q) mice present abnormalities in the retina as in patients: ataxin 7-positive neuronal intranuclear inclusions that were reduced by interferon beta treatment. Finally, since neuronal death does not occur in the cerebellum of SCA7(266Q/5Q) mice, we showed in primary cell cultures expressing mutant ataxin 7 that interferon beta treatment improves Purkinje cell survival.

Alteration of fatty-acid-metabolizing enzymes affects mitochondrial form and function in hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.

KIF1A missense mutations in SPG30, an autosomal recessive spastic paraplegia: distinct phenotypes according to the nature of the mutations.

The hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases characterised by progressive spasticity in the lower limbs. The nosology of autosomal recessive forms is complex as most mapped loci have been identified in only one or a few families and account for only a small percentage of patients. We used next-generation sequencing focused on the SPG30 chromosomal region on chromosome 2q37.3 in two patients from the original linked family. In addition, wide genome scan and candidate gene analysis were performed in a second family of Palestinian origin. We identified a single homozygous mutation, p.R350G, that was found to cosegregate with the disease in the SPG30 kindred and was absent in 970 control chromosomes while affecting a strongly conserved amino acid at the end of the motor domain of KIF1A. Homozygosity and linkage mapping followed by mutation screening of KIF1A allowed us to identify a second mutation, p.A255V, in the second family. Comparison of the clinical features with the nature of the mutations of all reported KIF1A families, including those reported recently with hereditary sensory and autonomic neuropathy, suggests phenotype-genotype correlations that may help to understand the mechanisms involved in motor neuron degeneration. We have shown that mutations in the KIF1A gene are responsible for SPG30 in two autosomal recessive HSP families. In published families, the nature of the KIF1A mutations seems to be of good predictor of the underlying phenotype and vice versa.

Prokineticin 2 expression is associated with neural repair of injured adult zebrafish telencephalon.

Prokineticin 2 (PROK2) is a secreted protein that regulates diverse biological processes including olfactory bulb neurogenesis in adult mammals. However, its precise role in this process is as yet not fully understood. Because it is well known that adult teleost fish, including zebrafish, display an intense proliferative activity in several brain regions, we took advantage of this feature to analyze the distribution of PROK2 transcripts in the adult zebrafish brain and during injury-induced telencephalon (TC) regeneration. First, we characterized the zebrafish PROK2 gene and showed that its transcription takes place in almost all proliferating areas previously identified in adult zebrafish brain. Moreover, in TC, PROK2 transcription was mainly restricted to neurons. Next, using a novel model of TC injury in adult zebrafish, we observed that TC lesion induced a dramatic increase in cell proliferation within the injured hemisphere in regions located both adjacent and distal to injury sites. Moreover, our data strongly suggest that cell proliferation was followed by the migration of newly generated neurons toward injury sites. In addition, we observed a transient over-expression of PROK2 transcripts, which was detected in cells surrounding the lesion during the very first days post injury, and, a few days later, in broad cell rows extending from cortical regions of the TC toward injury sites. PROK2 over-expression was no longer detected when the regeneration process was close to completion, showing that ectopic PROK2 transcription paralleled neuronal regeneration. Taken together, our results suggest that in adult zebrafish brain, PROK2 may play a role in both constitutive and injury-induced neurogenesis.

Developmental cell death is enhanced in the cerebral cortex of mice lacking the brain vesicular monoamine transporter.

Neurotransmitters have emerged as important players in the control of programmed cell death in the cerebral cortex. We report that genetic depletion of serotonin, dopamine, and norepinephrine in mice lacking the vesicular monoamine transporter (VMAT2 KO mice) causes an increase in cell death in the superficial layers of the cingulate and retrosplenial cortices during early postnatal life (postnatal days 0-4). Electron microscopy and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling indicated that this represents a form of apoptosis. Caspase-3 and -9 are over activated in the VMAT2 KO cortex and Bcl-X(L) is downregulated, whereas the apoptosis-inducing factor caspase-8 and FasL/FasR pathway are not involved. Partial inhibition of serotonin or/and catecholamine synthesis by pharmacological treatments or genetic reduction of serotonin neuron number in mice lacking the transcription factor Pet-1 (pheochromocytoma 12 E26 transformation-specific) did not modify the cell death ratios in the cerebral cortex. However, when monoamine oxidase type A was invalidated in the VMAT2 KO background (VMAT2-MAOA DKO mice), increases in 5-HT levels coincided with a reduction of cell death and a normalization of Bcl-X(L) expression. trkB signaling is not implicated in the anti-apoptotic effects of MAOA inhibition because BDNF mRNA levels were unchanged in VMAT2-MAOA DKO mice and because the massive cell death in the cerebral cortex of trkB KO mice is also reverted by genetic invalidation of the MAOA gene. Finally the broad 5-HT2 receptor agonist (-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride prevented the increase in cell death of VMAT2 KO mice. Altogether, these results suggest that high levels of serotonin, acting through 5-HT2 receptors, have neuroprotective action on cortical neurons by controlling Bcl-X(L) mRNA levels and that this action is independent of trkB signaling.

Polyglutamine and polyalanine expansions in ataxin7 result in different types of aggregation and levels of toxicity.

Spinocerebellar ataxia type 7 (SCA7) is caused by expansion of a (CAG)n repeat in the ataxin7 gene, resulting in an abnormally long polyglutamine polyQ tract in the translated protein that aggregates in the form of neuronal intranuclear inclusions. Polyalanine (polyA) stretches, implicated in several genetic disorders, also appear to aggregate. To investigate the role of the aggregates in the pathologies, we compared the effects of ataxin7 containing a polyA (ataxin7 - 90A) or polyQ (ataxin7 - 100Q) expansion in HEK 293 cells and in primary cultures of rat mesencephalon. Both proteins formed nuclear and perinuclear aggregates that contained molecular chaperones and components of the ubiquitin-proteasome system, suggesting that they were abnormally folded. Ataxin-90A aggregates differed morphologically from ataxin7 - 100Q aggregates, consisted of small and amorphous rather than fibrillar inclusions and were more toxic to mesencephalic neurons, suggesting that toxicity was determined by the type of aggregate rather than the cellular misfolding response.

Behavioral disorder, dementia, ataxia, and rigidity in a large family with TATA box-binding protein mutation.

BACKGROUND: Spinocerebellar ataxia type 17 is an autosomal dominant cerebellar ataxia caused by a CAG repeat expansion in the TATA box-binding protein gene. Ataxia is typically the first sign whereas behavioral symptoms occur later. OBJECTIVE: To characterize the unusual phenotypic expression of a large spinocerebellar ataxia type 17 kindred. DESIGN: Clinical, neuropathological, and molecular genetic characterization of a 4-generation family with 16 affected patients. RESULTS: Behavioral symptoms and frontal impairment dominated the early stages preceding ataxia, rigidity, and dystonic movements. Neuropathological examination showed cortical, subcortical, and cerebellar atrophy. Purkinje cell loss and gliosis, pseudohypertrophic degeneration of the inferior olive, marked neuronal loss and gliosis in the caudate nucleus, and in the medial thalamic nuclei were salient features together with neuronal intranuclear inclusions stained with anti-TATA box-binding protein and antipolyglutamine antibodies. The disease was caused by a stable 52 CAG repeat expansion of the TATA box-binding protein gene, although there was apparent variability in the age of onset. CONCLUSION: The characteristics of this family broaden the clinical picture of spinocerebellar ataxia type 17: initial presenile dementia with behavioral symptoms should be added to ataxia, rigidity, and dystonic movements, which are more commonly encountered.

PML nuclear bodies and neuronal intranuclear inclusion in polyglutamine diseases.

In polyglutamine diseases, accumulation in the nucleus of mutant proteins induces the formation of neuronal intranuclear inclusions (NIIs). The nucleus is compartmentalized into structural and functional domains, which are involved in NII formation. Promyelocytic leukemia protein (PML), a major component of nuclear bodies, and mSin3A, a component of the transcription co-repressor complex, were used to investigate how the intranuclear domains/sites relate to NII formation in SCA2, SCA3, SCA7, SCA17 and DRPLA brains. We demonstrate that the size of PML-positive intranuclear structures was larger in pathological brains than in control ones and that these structures contained mutant proteins. PML colocalized only with small NIIs, which maintained the ring-like structure of normal nuclear bodies. Enlarged ring-like PML-positive structures, devoid of mutant proteins, were also found and might represent structures where mutant polyglutamine proteins have been successfully processed. These data suggest that NIIs originate from nuclear bodies, where mutant proteins accumulate for degradation.

Two populations of neuronal intranuclear inclusions in SCA7 differ in size and promyelocytic leukaemia protein content.

Spinocerebellar ataxia type 7 (SCA7) is a hereditary progressive cerebellar ataxia with retinal degeneration associated with an abnormally expanded polyglutamine stretch. Neuronal intranuclear inclusions (NIIs), as in other polyglutamine diseases, are pathological hallmarks of these disorders. NIIs in polyglutamine diseases contain not only the protein with the expanded polyglutamine stretch but also other types of proteins. Several chaperone proteins related to the ubiquitin proteasome pathway, transcription factors and nuclear matrix proteins have been detected in NIIs. The composition of NIIs might reflect the process of NII formation and part of the pathogenesis of these diseases. To investigate how these proteins relate to the pathogenesis of SCA7, we performed immunohistochemical analyses of the composition of NIIs in two cases of SCA7. We demonstrated that there are two types of NIIs in SCA7 that differ in size and immunoreactivity to promyelocytic leukaemia protein (PML), one of the essential components of nuclear bodies (NBs; also called PML oncogenic domains). Small and large NIIs contained ataxin-7, human DnaJ homologue 2 (HDJ-2) and proteasome subunit 19S. In contrast, PML was found only in small NIIs. CREB-binding protein (CBP), another component of NBs, was distributed like PML in NIIs. Our results suggest that NIIs are formed by the accumulation of ataxin-7 in NBs, which become enlarged as they recruit related proteins.