Direct and Indirect Effects of Filamin A on Tau Pathology in Neuronal Cells.

In Alzheimer disease (AD), Tau, an axonal microtubule-associated protein, becomes hyperphosphorylated, detaches from microtubules, accumulates, and self-aggregates in the somatodendritic (SD) compartment. The accumulation of hyperphosphorylated and aggregated Tau is also seen in other neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD-Tau). Previous studies reported a link between filamin A (FLNA), an actin-binding protein found in the SD compartment, and Tau pathology. In the present study, we further explored this link. We confirmed the interaction of Tau with FLNA in neuroblastoma 2a (N2a) cells. This interaction was mediated by a domain located between the 157 and 383 amino acids (a.a.) of Tau. Our results also revealed that the overexpression of FLNA resulted in an intracellular accumulation of wild-type Tau and Tau mutants (P301L, V337M, and R406W) in N2a cells. Tau phosphorylation and cleavage by caspase-3 but not its aggregation were increased upon FLNA overexpression in N2a cells. In the parietal cortex of AD brain, insoluble FLNA was increased compared to control brain, but it did not correlate with Tau pathology. Interestingly, Tau binding to microtubules and F-actin was preserved upon FLNA overexpression in N2a cells. Lastly, our results revealed that FLNA also induced the accumulation of annexin A2, a Tau interacting partner involved in its axonal localization. Collectively, our data indicated that in Tauopathies, FLNA could contribute to Tau pathology by acting on Tau and annexin A2.

Unconventional secretion of tau by VAMP8 impacts its intra- and extracellular cleavage.

In Alzheimer's disease, Tau, a microtubule-associated protein, becomes hyperphosphorylated, detaches from microtubules, and accumulates in the somato-dendritic compartment where it forms insoluble aggregates. Tau also accumulates in the CSF of patients indicating that it is released by neurons. Consistent with this, several laboratories including ours have shown that Tau is secreted by neurons through unconventional secretory pathways. Recently, we reported that VAMP8, an R-SNARE found on late endosomes, increased Tau secretion and that secreted Tau was cleaved at the C-terminal. In the present study, we examined whether the increase of Tau secretion by VAMP8 affected its intra- and extracellular cleavage. Upon VAMP8 overexpression, an increase of Tau cleaved by caspase-3 in the cell lysate and medium was observed. This was correlated to an increase of active caspase-3 in the cell lysate and medium. Using a Tau mutant not cleavable by caspase-3, we demonstrated that Tau cleavage by caspase-3 was not necessary for its secretion upon VAMP8 overexpression. By adding recombinant Tau to the culture medium, we demonstrated that extracellular Tau cleavage by caspase-3 could occur because of the release of active caspase-3, which was the highest when VAMP8 was overexpressed. When cleavage of Tau by caspase-3 was prevented by using a non-cleavable mutant, secreted Tau was still cleaved at the C-terminal, the asparagine N410 contributing to it. Lastly, we demonstrated that N-terminal of Tau regulated the secretion pattern of a Tau fragment containing the microtubule-binding domain and the C-terminal of Tau upon VAMP8 overexpression. Collectively, the above observations indicate that VAMP8 overexpression affects the intra- and extracellular cleavage pattern of Tau.

Clearance of intracellular tau protein from neuronal cells via VAMP8-induced secretion.

In Alzheimer's disease (AD), tau, a microtubule-associated protein (MAP), becomes hyperphosphorylated, aggregates, and accumulates in the somato-dendritic compartment of neurons. In parallel to its intracellular accumulation in AD, tau is also released in the extracellular space, as revealed by its increased presence in cerebrospinal fluid (CSF). Consistent with this, recent studies, including ours, have reported that neurons secrete tau, and several therapeutic strategies aim to prevent the intracellular tau accumulation. Previously, we reported that late endosomes were implicated in tau secretion. Here, we explore the possibility of preventing intracellular tau accumulation by increasing tau secretion. Using neuronal models, we investigated whether overexpression of the vesicle-associated membrane protein 8 (VAMP8), an R-SNARE found on late endosomes, could increase tau secretion. The overexpression of VAMP8 significantly increased tau secretion, decreasing its intracellular levels in the neuroblastoma (N2a) cell line. Increased tau secretion by VAMP8 was also observed in murine hippocampal slices. The intracellular reduction of tau by VAMP8 overexpression correlated to a decrease of acetylated tubulin induced by tau overexpression in N2a cells. VAMP8 staining was preferentially found on late endosomes in N2a cells. Using total internal reflection fluorescence (TIRF) microscopy, the fusion of VAMP8-positive vesicles with the plasma membrane was correlated to the depletion of tau in the cytoplasm. Finally, overexpression of VAMP8 reduced the intracellular accumulation of tau mutants linked to frontotemporal dementia with parkinsonism and α-synuclein by increasing their secretion. Collectively, the present data indicate that VAMP8 could be used to increase tau and α-synuclein clearance to prevent their intracellular accumulation.

Plasma oxysterols: biomarkers for diagnosis and treatment in spastic paraplegia type 5.

The hereditary spastic paraplegias are an expanding and heterogeneous group of disorders characterized by spasticity in the lower limbs. Plasma biomarkers are needed to guide the genetic testing of spastic paraplegia. Spastic paraplegia type 5 (SPG5) is an autosomal recessive spastic paraplegia due to mutations in CYP7B1, which encodes a cytochrome P450 7α-hydroxylase implicated in cholesterol and bile acids metabolism. We developed a method based on ultra-performance liquid chromatography electrospray tandem mass spectrometry to validate two plasma 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC) as diagnostic biomarkers in a cohort of 21 patients with SPG5. For 14 patients, SPG5 was initially suspected on the basis of genetic analysis, and then confirmed by increased plasma 25-OHC, 27-OHC and their ratio to total cholesterol. For seven patients, the diagnosis was initially based on elevated plasma oxysterol levels and confirmed by the identification of two causal CYP7B1 mutations. The receiver operating characteristic curves analysis showed that 25-OHC, 27-OHC and their ratio to total cholesterol discriminated between SPG5 patients and healthy controls with 100% sensitivity and specificity. Taking advantage of the robustness of these plasma oxysterols, we then conducted a phase II therapeutic trial in 12 patients and tested whether candidate molecules (atorvastatin, chenodeoxycholic acid and resveratrol) can lower plasma oxysterols and improve bile acids profile. The trial consisted of a three-period, three-treatment crossover study and the six different sequences of three treatments were randomized. Using a linear mixed effect regression model with a random intercept, we observed that atorvastatin decreased moderately plasma 27-OHC (∼30%, P < 0.001) but did not change 27-OHC to total cholesterol ratio or 25-OHC levels. We also found an abnormal bile acids profile in SPG5 patients, with significantly decreased total serum bile acids associated with a relative decrease of ursodeoxycholic and lithocholic acids compared to deoxycholic acid. Treatment with chenodeoxycholic acid restored bile acids profile in SPG5 patients. Therefore, the combination of atorvastatin and chenodeoxycholic acid may be worth considering for the treatment of SPG5 patients but the neurological benefit of these metabolic interventions remains to be evaluated in phase III therapeutic trials using clinical, imaging and/or electrophysiological outcome measures with sufficient effect sizes. Overall, our study indicates that plasma 25-OHC and 27-OHC are robust diagnostic biomarkers of SPG5 and shall be used as first-line investigations in any patient with unexplained spastic paraplegia.

New practical definitions for the diagnosis of autosomal recessive spastic ataxia of Charlevoix-Saguenay.

OBJECTIVE: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in the SACS gene. SACS encodes sacsin, a protein whose function remains unknown, despite the description of numerous protein domains and the recent focus on its potential role in the regulation of mitochondrial physiology. This study aimed to identify new mutations in a large population of ataxic patients and to functionally analyze their cellular effects in the mitochondrial compartment. METHODS: A total of 321 index patients with spastic ataxia selected from the SPATAX network were analyzed by direct sequencing of the SACS gene, and 156 patients from the ATAXIC project presenting with congenital ataxia were investigated either by targeted or whole exome sequencing. For functional analyses, primary cultures of fibroblasts were obtained from 11 patients carrying either mono- or biallelic variants, including 1 case harboring a large deletion encompassing the entire SACS gene. RESULTS: We identified biallelic SACS variants in 33 patients from SPATAX, and in 5 nonprogressive ataxia patients from ATAXIC. Moreover, a drastic and recurrent alteration of the mitochondrial network was observed in 10 of the 11 patients tested. INTERPRETATION: Our results permit extension of the clinical and mutational spectrum of ARSACS patients. Moreover, we suggest that the observed mitochondrial network anomalies could be used as a trait biomarker for the diagnosis of ARSACS when SACS molecular results are difficult to interpret (ie, missense variants and heterozygous truncating variant). Based on our findings, we propose new diagnostic definitions for ARSACS using clinical, genetic, and cellular criteria.

REEP1 mutations in SPG31: frequency, mutational spectrum, and potential association with mitochondrial morpho-functional dysfunction.

Hereditary spastic paraplegias (HSP) constitute a heterogeneous group of neurodegenerative disorders characterized at least by slowly progressive spasticity of the lower limbs. Mutations in REEP1 were recently associated with a pure dominant HSP, SPG31. We sequenced all exons of REEP1 and searched for rearrangements by multiplex ligation-dependent probe amplification (MLPA) in a large panel of 175 unrelated HSP index patients from kindreds with dominant inheritance (AD-HSP), with either pure (n = 102) or complicated (n = 73) forms of the disease, after exclusion of other known HSP genes. We identified 12 different heterozygous mutations, including two exon deletions, associated with either a pure or a complex phenotype. The overall mutation rate in our clinically heterogeneous sample was 4.5% in French families with AD-HSP. The phenotype was restricted to pyramidal signs in the lower limbs in most patients but nine had a complex phenotype associating axonal peripheral neuropathy (= 5/11 patients) including a Silver-like syndrome in one patient, and less frequently cerebellar ataxia, tremor, dementia. Interestingly, we evidenced abnormal mitochondrial network organization in fibroblasts of one patient in addition to defective mitochondrial energy production in both fibroblasts and muscle, but whether these anomalies are directly or indirectly related to the mutations remains uncertain.