Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

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.

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.

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.

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.

Cellular distribution and subcellular localization of spatacsin and spastizin, two proteins involved in hereditary spastic paraplegia.

Truncating mutations in the SPG11 and SPG15 genes cause complicated spastic paraplegia, severe neurological conditions due to loss of the functions of spatacsin and spastizin, respectively. We developed specific polyclonal anti-spatacsin (SPG11) and anti-spastizin (SPG15) antisera, which we then used to explore the intracellular and tissue localizations of these proteins. We observed expression of both proteins in human and rat central nervous system, which was particularly strong in cortical and spinal motor neurons as well as in retina. Both proteins were also expressed ubiquitously and strongly in embryos. In cultured cells, these two proteins had similar diffuse punctate, cytoplasmic and sometimes nuclear (spastizin) distributions. They partially co-localized with multiple organelles, particularly with protein-trafficking vesicles, endoplasmic reticulum and microtubules. Spastizin was also found at the mitochondria surface. This first study of the endogenous expression of spatacsin and spastizin shows similarities in their expression patterns that could account for their overlapping clinical phenotypes and involvement in a common protein complex.

Subcellular topography of neuronal Abeta peptide in APPxPS1 transgenic mice.

In transgenic mice expressing human mutant beta-amyloid precursor protein (APP) and mutant presenilin-1 (PS1), Abeta antibodies labeled granules, about 1 microm in diameter, in the perikaryon of neurons clustered in the isocortex, hippocampus, amygdala, thalamus, and brainstem. The granules were present before the onset of Abeta deposits; their number increased up to 9 months and decreased in 15-month-old animals. They were immunostained by antibodies against Abeta 40, Abeta 42, and APP C-terminal region. In double immunofluorescence experiments, the intracellular Abeta co-localized with lysosome markers and less frequently with MG160, a Golgi marker. Abeta accumulation correlated with an increased volume of lysosomes and Golgi apparatus, while the volume of endoplasmic reticulum and early endosomes did not change. Some granules were immunolabeled with an antibody against flotillin-1, a raft marker. At electron microscopy, Abeta, APP-C terminal, cathepsin D, and flotillin-1 epitopes were found in the lumen of multivesicular bodies. This study shows that Abeta peptide and APP C-terminal region accumulate in multivesicular bodies containing lysosomal enzymes, while APP N-terminus is excluded from them. Multivesicular bodies could secondarily liberate their content in the extracellular space as suggested by the association of cathepsin D with Abeta peptide in the extracellular space.

Amyotrophic lateral sclerosis with neuronal intranuclear protein inclusions.

A 46-year-old patient developed amyotrophic lateral sclerosis (ALS) characterized by rapid progression. She needed respiratory assistance after a course of 9 months. She died 4.5 years after onset. Autopsy showed dramatic atrophy of the spinal cord, sparing only the posterior tracts, associated with neuronal loss and astrogliosis in various areas including the anterior horns, motor cortex, striatum, thalamus, and substantia nigra. Ubiquitin immunohistochemistry showed rare skein-like inclusions in the surviving spinal and medullary motor neurons. Eosinophilic inclusions were found in the nuclei of pyramidal neurons in the hippocampus. These inclusions were immunoreactive to antibodies against ubiquitin, promyelocytic leukemia gene product, proteasome, and ataxin-3. They were not immunoreactive to antibodies against tau, cystatin C, neurofilament, alpha-synuclein, SOD-1, and polyglutamine (1C2), and were not stained by ethidium bromide. Similar inclusions were found in the motor cortex. The immunoreactivity of the inclusions was similar to that encountered in diseases associated with CAG repeats, except for the negativity of the immunolabelling with 1C2. At the ultrastructural level, the nuclear inclusions were made of straight filaments (10-12 nm in diameter) arranged at random, reminiscent of the polyglutamine intranuclear hyaline inclusions.