Golgi localization of SARS-CoV-2 spike protein and interaction with furin in cerebral COVID-19 microangiopathy: a clue to the central nervous system involvement?

In a neuropathological series of 20 COVID-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by MRI. The cases presented with microhemorrhages evocative of small artery diseases. This COVID-19 associated cerebral microangiopathy (CCM) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. There was evidence of blood-brain-barrier leakage. Fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. While no viral particle or viral RNA was found in the brain, the SARS-CoV-2 spike protein was detected in the Golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. Endothelial cells in culture were not permissive to SARS-CoV-2 replication. The distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. In the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. In contrast, in cerebral endothelial cells the excretion cycle was blocked in the Golgi apparatus. Interruption of the excretion cycle could explain the difficulty of SARS-CoV-2 to infect endothelial cells in vitro and to produce viral RNA in the brain. Specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of COVID-19 associated cerebral microangiopathy. Furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy.

CNS-associated T-lymphocytes in a mouse model of Hereditary Spastic Paraplegia type 11 (SPG11) are therapeutic targets for established immunomodulators.

Pharmacological targeting of neuroinflammation in distinct models of genetically mediated disorders of the central nervous system (CNS) has been shown to attenuate disease outcome significantly. These include mouse models mimicking distinct subtypes of neuronal ceroid lipofuscinoses (NCL, CLN diseases) as well as hereditary spastic paraplegia type 2 (HSP/SPG2). We here show in a model of another, complicated HSP form (SPG11) that there is neuroinflammation in distinct compartments of the diseased CNS. Using a proof-of-principle experiment, we provide evidence that genetically targeting the adaptive immune system dampens disease progression including gait disturbance, demonstrating a pathogenic impact of neuroinflammation. Translating these studies into a clinically applicable approach, we show that the established immunomodulators fingolimod and teriflunomide significantly attenuate the neurodegenerative phenotype and improve gait performance in the SPG11 model, even when applied relatively late during disease progression. Particularly abnormalities in gait coordination, representing ataxia, could be attenuated, while features indicative of reduced strength during walking did not respond to treatment. Our study identifies neuroinflammation by the adaptive immune system as a robust and targetable disease amplifier in a mouse model of SPG11 and may thus pave the way for a translational approach in humans implicating approved immunomodulators.

Alterations of Neuronal Lysosomes in Alzheimer's Disease and in APPxPS1-KI Mice.

BACKGROUND: The cellular and molecular alterations associated with synapse and neuron loss in Alzheimer's disease (AD) remain unclear. In transgenic mouse models that express mutations responsible for familial AD, neuronal and synaptic losses occur in populations that accumulate fibrillar amyloid-ß 42 (Aß42) intracellularly. OBJECTIVE: We aimed to study the subcellular localization of these fibrillar accumulations and whether such intraneuronal assemblies could be observed in the human pathology. METHODS: We used immunolabeling and various electron microscopy techniques on APP x presenilin1 - knock-in mice and on human cortical biopsies and postmortem samples. RESULTS: We found an accumulation of Aß fibrils in lipofuscin granule-like organelles in APP x presenilin1 - knock-in mice. Electron microscopy of human cortical biopsies also showed an accumulation of undigested material in enlarged lipofuscin granules in neurons from AD compared to age-matched non-AD patients. However, in those biopsies or in postmortem samples we could not detect intraneuronal accumulations of Aß fibrils, neither in the lipofuscin granules nor in other intraneuronal compartments. CONCLUSION: The intralysosomal accumulation of Aß fibrils in specific neuronal populations in APPxPS1-KI mice likely results from a high concentration of Aß42 in the endosome-lysosome system due to the high expression of the transgene in these neurons.

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.

Loss of spatacsin impairs cholesterol trafficking and calcium homeostasis.

Mutations in SPG11, leading to loss of spatacsin function, impair the formation of membrane tubules in lysosomes and cause lysosomal lipid accumulation. However, the full nature of lipids accumulating in lysosomes and the physiological consequences of such accumulation are unknown. Here we show that loss of spatacsin inhibits the formation of tubules on lysosomes and prevents the clearance of cholesterol from this subcellular compartment. Accumulation of cholesterol in lysosomes decreases cholesterol levels in the plasma membrane, enhancing the entry of extracellular calcium by store-operated calcium entry and increasing resting cytosolic calcium levels. Higher cytosolic calcium levels promote the nuclear translocation of the master regulator of lysosomes TFEB, preventing the formation of tubules and the clearance of cholesterol from lysosomes. Our work reveals a homeostatic balance between cholesterol trafficking and cytosolic calcium levels and shows that loss of spatacsin impairs this homeostatic equilibrium.

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination.

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides that Contribute to Neurodegeneration.

Lysosome membrane recycling occurs at the end of the autophagic pathway and requires proteins that are mostly encoded by genes mutated in neurodegenerative diseases. However, its implication in neuronal death is still unclear. Here, we show that spatacsin, which is required for lysosome recycling and whose loss of function leads to hereditary spastic paraplegia 11 (SPG11), promotes clearance of gangliosides from lysosomes in mouse and human SPG11 models. We demonstrate that spatacsin acts downstream of clathrin and recruits dynamin to allow lysosome membrane recycling and clearance of gangliosides from lysosomes. Gangliosides contributed to the accumulation of autophagy markers in lysosomes and to neuronal death. In contrast, decreasing ganglioside synthesis prevented neurodegeneration and improved motor phenotype in a SPG11 zebrafish model. Our work reveals how inhibition of lysosome membrane recycling leads to the deleterious accumulation of gangliosides, linking lysosome recycling to neurodegeneration.

Evidence for altered dendritic spine compartmentalization in Alzheimer's disease and functional effects in a mouse model.

Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allowed us to precisely measure spine parameters. Mathematical modeling indicated that the shortening and widening of the necks should alter the electrical compartmentalization of the spines, leading to reduced postsynaptic potentials in spine heads, but not in soma. Accordingly, there was no alteration in basal synaptic transmission, but long-term potentiation and spatial memory were impaired. These results indicate that an alteration of spine morphology could be involved in the early cognitive deficits associated with AD.

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.

Loss of spatacsin function alters lysosomal lipid clearance leading to upper and lower motor neuron degeneration.

Mutations in SPG11 account for the most common form of autosomal recessive hereditary spastic paraplegia (HSP), characterized by a gait disorder associated with various brain alterations. Mutations in the same gene are also responsible for rare forms of Charcot-Marie-Tooth (CMT) disease and progressive juvenile-onset amyotrophic lateral sclerosis (ALS). To elucidate the physiopathological mechanisms underlying these human pathologies, we disrupted the Spg11 gene in mice by inserting stop codons in exon 32, mimicking the most frequent mutations found in patients. The Spg11 knockout mouse developed early-onset motor impairment and cognitive deficits. These behavioral deficits were associated with progressive brain atrophy with the loss of neurons in the primary motor cortex, cerebellum and hippocampus, as well as with accumulation of dystrophic axons in the corticospinal tract. Spinal motor neurons also degenerated and this was accompanied by fragmentation of neuromuscular junctions and muscle atrophy. This new Spg11 knockout mouse therefore recapitulates the full range of symptoms associated with SPG11 mutations observed in HSP, ALS and CMT patients. Examination of the cellular alterations observed in this model suggests that the loss of spatacsin leads to the accumulation of lipids in lysosomes by perturbing their clearance from these organelles. Altogether, our results link lysosomal dysfunction and lipid metabolism to neurodegeneration and pinpoint a critical role of spatacsin in lipid turnover.

Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins.

BACKGROUND: We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure. RESULTS: LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7. CONCLUSIONS: This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects.

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.

The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.

There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.

Role of the phosphoinositide phosphatase FIG4 gene in familial epilepsy with polymicrogyria.

OBJECTIVE: The aim of this study was to identify the causal gene in a consanguineous Moroccan family with temporo-occipital polymicrogyria, psychiatric manifestations, and epilepsy, previously mapped to the 6q16-q22 region. METHODS: We used exome sequencing and analyzed candidate variants in the 6q16-q22 locus, as well as a rescue assay in Fig4-null mouse fibroblasts and immunohistochemistry of Fig4-null mouse brains. RESULTS: A homozygous missense mutation (p.Asp783Val) in the phosphoinositide phosphatase gene FIG4 was identified. Pathogenicity of the variant was supported by impaired rescue of the enlarged vacuoles in transfected fibroblasts from Fig4-deficient mice. Histologic examination of Fig4-null mouse brain revealed neurodevelopmental impairment in the hippocampus, cortex, and cerebellum as well as impaired cerebellar gyration/foliation reminiscent of human cortical malformations. CONCLUSIONS: This study extends the spectrum of phenotypes associated with FIG4 mutations to include cortical malformation associated with seizures and psychiatric manifestations, in addition to the previously described Charcot-Marie-Tooth disease type 4J and Yunis-Varón syndrome.

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.

Juvenile frontotemporal dementia with parkinsonism associated with tau mutation G389R.

Frontotemporal lobe degeneration includes a large spectrum of neurodegenerative disorders. Patients with frontotemporal dementia with parkinsonism linked to chromosome 17 exhibit heterogeneity in both clinical and neuropathological features. Here, we report the case of a young patient with a G389R mutation. This teenager girl was 17 years old when she progressively developed severe behavioral disturbances. First, she was considered to be suffering from atypical depression. After 2 years, she was referred to the department of neurology. By this time, the patient exhibited typical frontotemporal dementia with mild extrapyramidal disorders. The main behavioral features included apathy and reduced speech output. MRI and SPECT showed a frontotemporal atrophy and hypofixation, respectively. She died 7 years after onset. Three relatives on her father side had also died after early onset dementia. Genetic testing revealed a heterozygous guanine to cytosine mutation at the first base of codon 389 (Exon 13) of MAPT, the tau gene, resulting in a glycine to arginine substitution, in the patient and her non-affected father. Postmortem neuropathological and biochemical data indicate a Pick-like tau pathology but with phosphoserine 262-positive immunoreactivity. This case is remarkable because of the extremely early onset of the disease.

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.