Lysosome dysfunction has been widely implicated in many models of neurodegeneration, but much less is understood of its involvement during brain development in health and disease. Hereditary spastic paraplegia caused by mutations in the SPG11 gene is a neurodegenerative disorder characterized by lysosome dysfunction, which also presents neurodevelopmental alterations. Using knockout mouse and cortical organoid models derived from induced pluripotent stem cells, we show that lysosome dysfunction caused by SPG11 mutations decreases the proliferation of neural progenitor cells at early stages of cortical development. At the cellular level, SPG11 mutations cause accumulation of calcium in lysosomes, which reduces proliferation of neural progenitor cells and diminishes apical tight junctions. RNA sequencing analysis revealed that these phenotypes in SPG11 organoids are caused by hypoactivation of mammalian target of rapamycin (mTOR) signaling. The latter is a consequence of lysosomal recruitment of the enzyme PI4K2A (phosphatidylinositol 4-kinase type 2 alpha) resulting in higher levels of its product PI(4)P (phosphatidylinositol-4-phosphate), a described regulator of the mTOR pathway. Pharmacological modulation of the function of the lysosomal calcium channel TRPML1 successfully corrected all developmental phenotypes in cortical organoids, highlighting the critical role of lysosomal calcium in signaling during the early phase of cortical development.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are part of a well-established disease continuum, underpinned by TDP43-pathology. In contrast, the clinical manifestations of Tau-linked disorders are typically limited to cognitive phenotypes or atypical parkinsonism, although few reports describe motor neuron involvement associated with MAPT (microtubule-associated protein Tau) mutations. This study aimed to investigate the contribution of MAPT to the ALS phenotype. We analyzed a whole-exome sequencing database comprising 470 ALS patients and explored the pathogenicity of the identified variants through familial, clinical, neuropathological, and cellular studies. We identified two missense variants in the Tau repeat domains: the novel p.I308T variant, in a patient with early-onset ALS, and the p.P364S mutation in three families with spinal- or respiratory-onset ALS. Segregation of this mutation with disease could be confirmed in two affected cousins. The observation of p.P364S patient’s tissue showed accumulations of hyperphosphorylated Tau in various brain regions, prominent in the motor cortex with Lewy body-like inclusions, along with a C-terminal cleaved form of Tau in muscle. In NSC-34 motor neuron cells expressing p.I308T or p.P364S mutants, Tau was discontinuous along the neurites, with clusters of mitochondria resulting from impaired mitochondrial motility. These findings expand the molecular understanding of ALS to include MAPT mutations. MAPT analysis should be incorporated into ALS genetic screening, particularly in patients with a familial history of the disease. Recognizing the full spectrum of MAPT-linked neurodegenerative diseases is of considerable interest, given the ongoing efforts to develop MAPT-targeted therapies.

We have previously demonstrated that neuroinflammation by the adaptive immune system acts as a robust and targetable disease amplifier in a mouse model of Spastic Paraplegia, type 11 (SPG11), a complicated form of Hereditary Spastic Paraplegia (HSP). While we identified an impact of neuroinflammation on distinct neuropathological changes and gait performance, neuropsychological features, typical and clinically highly relevant symptoms of complicated HSPs, were not addressed. Here we show that the corresponding SPG11 mouse model shows distinct behavioral abnormalities, particularly related to social behavior thus partially reflecting the neuropsychological changes in patients. We provide evidence that some behavioral abnormalities can be mitigated by genetic inactivation of the adaptive immune system. Translating this into a clinically applicable approach, we show that treatment with the established immunomodulators fingolimod or teriflunomide significantly attenuates distinct behavioral abnormalities, with the most striking effect on social behavior. This study links neuroinflammation to behavioral abnormalities in a mouse model of SPG11 and may thus pave the way for using immunomodulators as a treatment approach for SPG11 and possibly other complicated forms of HSP with neuropsychological involvement.

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.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

Hereditary spastic paraplegias consist of a group of rare neurodegenerative diseases characterized by lower limb spasticity. These inherited Mendelian disorders show high genetic variability associated with wide clinical diversity. Pathophysiological investigations have suggested that mutations in genes affecting the same cellular pathway generally lead to similar clinical symptoms, highlighting the importance of genetic mutation in these diseases. However, phenotype-genotype correlations have failed to explain the observed large inter-individual variability linked to mutations in a single gene, suggesting that genetics alone is not sufficient to explain symptom diversity. The identification of biomarkers, such as neurofilament light chain, could fill the gap and predict disease evolution.

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.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movement of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, we demonstrate that spatacsin is an ER-resident protein that regulates ER-lysosomes contacts to promote lysosome motility, shown by the presence of tubular lysosomes. Tubular lysosomes, which are highly dynamic, are entangled in a network of tubular ER. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to lysosome trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150 Glued , respectively. Ultimately, investigations in polarized neurons demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

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 interferon-alpha levels as putative biomarkers in mouse and in 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 extra neuronal tissues.

Hereditary spastic paraplegias (HSPs) are a group of rare, inherited, neurological diseases characterized by broad clinical and genetic heterogeneity. Lower-limb spasticity with first motoneuron involvement is the core symptom of all HSPs. As spasticity is a syndrome and not a disease, it develops on top of other neurological signs (ataxia, dystonia, and parkinsonism). Indeed, the definition of genes responsible for HSPs goes beyond the 79 identified SPG genes. In order to avoid making a catalog of the different genes involved in HSP in any way, we have chosen to focus on the HSP with cerebellar ataxias since this is a frequent association described for several genes. This overlap leads to an intermediary group of spastic ataxias which is actively genetically and clinically studied. The most striking example is SPG7, which is responsible for HSP or cerebellar ataxia or both. There are no specific therapies against HSPs, and there is a dearth of randomized trials in patients with HSP, especially on spasticity when it likely results from other mechanisms. Thus far, no gene-specific therapy has been developed for HSP, but emerging therapies in animal models and neurons derived from induced pluripotent stem cells are potential treatments for patients.

Neurofilament light chain (NfL) is a marker of brain atrophy and predictor of disease progression in rare diseases such as Huntington Disease, but also in more common neurological disorders such as Alzheimer's disease. The aim of this study was to measure NfL longitudinally in autosomal dominant spinocerebellar ataxias (SCAs) and establish correlation with clinical and imaging parameters. We enrolled 62 pathological expansions carriers (17 SCA1, 13 SCA2, 19 SCA3, and 13 SCA7) and 19 age-matched controls in a prospective biomarker study between 2011 and 2015 and followed for 24 months at the Paris Brain Institute. We performed neurological examination, brain 3 T MRI and plasma NfL measurements using an ultrasensitive single-molecule array at baseline and at the two-year follow-up visit. We evaluated NfL correlations with ages, CAG repeat sizes, clinical scores and volumetric brain MRIs. NfL levels were significantly higher in SCAs than controls at both time points (p < 0.001). Age-adjusted NfL levels were significantly correlated at baseline with clinical scores (p < 0.01). We identified optimal NfL cut-off concentrations to differentiate controls from carriers for each genotype (SCA1 16.87 pg/mL, SCA2, 19.1 pg/mL, SCA3 16.04 pg/mL, SCA7 16.67 pg/mL). For all SCAs, NfL concentration was stable over two years (p = 0.95) despite a clinical progression (p < 0.0001). Clinical progression between baseline and follow-up was associated with higher NfL concentrations at baseline (p = 0.04). Of note, all premanifest carriers with NfL levels close to cut off concentrations had signs of the disease at follow-up. For all SCAs, the higher the observed NfL, the lower the pons volume at baseline (p < 0.01) and follow-up (p = 0.02). Higher NfL levels at baseline in all SCAs predicted a decrease in cerebellar volume (p = 0.03). This result remained significant for SCA2 only among all genotypes (p = 0.02). Overall, plasma NfL levels at baseline in SCA expansion carriers predict cerebellar volume change and clinical score progression. NfL levels might help refine inclusion criteria for clinical trials in carriers with very subtle signs.

Hereditary spastic paraplegias (HSP) are a group of neurodegenerative diseases sharing spasticity in lower limbs as common symptom. There is a large clinical variability in the presentation of patients, partly underlined by the large genetic heterogeneity, with more than 60 genes responsible for HSP. Despite this large heterogeneity, the proteins with known function are supposed to be involved in a limited number of cellular compartments such as shaping of the endoplasmic reticulum or endolysosomal function. Yet, it is difficult to understand why alteration of such different cellular compartments can lead to degeneration of the axons of cortical motor neurons. A common feature that has emerged over the last decade is the alteration of lipid metabolism in this group of pathologies. This was first revealed by the identification of mutations in genes encoding proteins that have or are supposed to have enzymatic activities on lipid substrates. However, it also appears that mutations in genes affecting endoplasmic reticulum, mitochondria or endolysosome function can lead to changes in lipid distribution or metabolism. The aim of this review is to discuss the role of lipid metabolism alterations in the physiopathology of HSP, to evaluate how such alterations contribute to neurodegenerative phenotypes and to understand how this knowledge can help develop therapeutic strategy for HSP.

Rare genetic diseases affect a limited number of patients, but their etiology is often known, facilitating the development of reliable animal models and giving the opportunity to investigate physiopathology. Lysosomal storage disorders are a group of rare diseases due to primary alteration of lysosome function. These diseases are often associated with neurological symptoms, which highlighted the importance of lysosome in neurodegeneration. Likewise, other groups of rare neurodegenerative diseases also present lysosomal alteration. Lysosomes fuse with autophagosomes and endosomes to allow the degradation of their content thanks to hydrolytic enzymes. It has emerged that alteration of the autophagy–lysosome pathway could play a critical role in neuronal death in many neurodegenerative diseases. Using a repertoire of selected rare neurodegenerative diseases, we highlight that a variety of alterations of the autophagy–lysosome pathway are associated with neuronal death. Yet, in most cases, it is still unclear why alteration of this pathway can lead to neurodegeneration.

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.

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.

Membrane fusion is a key event in exocytosis of neurotransmitters and hormones stored in intracellular vesicles. In this process, soluble N‐ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are essential components of the exocytotic molecular machinery, while lipids have been seen traditionally as structural elements. However, the so‐called signalling lipids, such as sphingosine and arachidonic acid, interact with SNAREs and directly modulate the frequency and mode of fusion events. Interestingly, recent work has proved that the sphingosine analogue FTY‐720, used in the treatment of multiple sclerosis, mimics the effects of signalling lipids. In the present Review, we discuss recent investigations suggesting that endogenous signalling lipids and synthetic analogues can modulate important physiological aspects of secretion, such as quantal release, vesicle recruitment into active sites, vesicle transport and even organelle fusion in the cytosol. Therefore, these compounds are far from being merely structural components of cellular membranes.

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.