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In amyotrophic lateral sclerosis (ALS) caused by SOD1 gene mutations, both cell-autonomous and noncell-autonomous mechanisms lead to the selective degeneration of motoneurons (MN). Here, we evaluate the therapeutic potential of gene therapy targeting mutated SOD1 in mature astrocytes using mice expressing the mutated SOD1

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208-H140 hydrogen bond (“H-latch”) between the α2-α3 and β2-α2 loops of PrPC. Importantly, GDL that suppress the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced nineteen individual PrPC variants to PrPC-deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrPC. K27 may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.

Bile acids (BAs) are signalling molecules that mediate various cellular responses in both physiological and pathological processes. Several studies report that BAs can be detected in the brain1, yet their physiological role in the central nervous system is still largely unknown. Here we show that postprandial BAs can reach the brain and activate a negative-feedback loop controlling satiety in response to physiological feeding via TGR5, a G-protein-coupled receptor activated by multiple conjugated and unconjugated BAs2 and an established regulator of peripheral metabolism3–8. Notably, peripheral or central administration of a BA mix or a TGR5-specific BA mimetic (INT-777) exerted an anorexigenic effect in wild-type mice, while whole-body, neuron-specific or agouti-related peptide neuronal TGR5 deletion caused a significant increase in food intake. Accordingly, orexigenic peptide expression and secretion were reduced after short-term TGR5 activation. In vitro studies demonstrated that activation of the Rho–ROCK–actin-remodelling pathway decreases orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) release in a TGR5-dependent manner. Taken together, these data identify a signalling cascade by which BAs exert acute effects at the transition between fasting and feeding and prime the switch towards satiety, unveiling a previously unrecognized role of physiological feedback mediated by BAs in the central nervous system. Bile acids are shown to enter the brain and regulate short-term reductions in food intake after a meal by inhibiting neuropeptide release from agouti-related peptide/neuropeptide Y neurons.

α-Synuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and α-synuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like α-synuclein, chaperones the SNARE complex assembly and controls neurotransmitter release. α-Synuclein can rescue neurodegeneration in CSPαKO mice. However, whether α-synuclein aggregation alters CSPα expression and function is unknown. Here we show that α-synuclein aggregation at the synapse is associated with a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with α-synuclein aggregates and in vivo reduces synaptic α-synuclein aggregates increasing monomeric α-synuclein and restoring normal dopamine release in 1-120hαSyn mice. These novel findings reveal a mechanism by which α-synuclein aggregation alters CSPα at the synapse, and show that CSPα rescues α-synuclein aggregation-related phenotype in 1-120hαSyn mice similar to the effect of α-synuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage Parkinson’s disease., CSPα and αsynuclein (αsyn) are involved in synaptic function and neurotransmitter release via parallel pathways. Caló et al. show that αsyn aggregation at the synapse is associated with a reduction in CSPα. An increase in CSPα reduces synaptic αsyn aggregates, rescuing impaired striatal dopamine release in αsyn transgenic mice.

Background & Aims Nonalcoholic fatty liver disease (NAFLD) is considered a health epidemic with potential devastating effects on the patients and the healthcare systems. Current preclinical models of NAFLD are invariably imperfect and generally take a long time to develop. A mouse model of survival motor neuron (SMN) depletion (Smn2B/- mice) was recently shown to develop significant hepatic steatosis in less than 2 weeks from birth. The rapid onset of fatty liver in Smn2B/- mice provides an opportunity to identify molecular markers of NAFLD. Here, we investigated whether Smn2B/- mice display typical features of NAFLD/nonalcoholic steatohepatitis (NASH). Methods Biochemical, histologic, electron microscopy, proteomic, and high-resolution respirometry were used. Results The Smn2B/- mice develop microvesicular steatohepatitis within 2 weeks, a feature prevented by AAV9-SMN gene therapy. Although fibrosis is not overtly apparent in histologic sections of the liver, there is molecular evidence of fibrogenesis and presence of stellate cell activation. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in hepatic dysfunction in protein output, complement, coagulation, iron homeostasis, and insulin-like growth factor-1 metabolism. The NAFLD phenotype is likely due to non-esterified fatty acid overload from peripheral lipolysis subsequent to hyperglucagonemia compounded by reduced muscle use and insulin resistance. Despite the low hepatic mitochondrial content, isolated mitochondria show enhanced β-oxidation, likely as a compensatory response, resulting in the production of reactive oxygen species. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight because of their associated neurological condition (spinal muscular atrophy) and develop hypoglycemia. Conclusions The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early-onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations., Graphical abstract

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the most common motoneuron diseases affecting adults and infants, respectively. ALS and SMA are both characterized by the selective degeneration of motoneurons. Although different in their genetic etiology, growing evidence indicate that they share molecular and cellular pathogenic signatures that constitute potential common therapeutic targets. We previously described a motoneuron-specific death pathway elicited by the Fas death receptor, whereby vulnerable ALS motoneurons show an exacerbated sensitivity to Fas activation. However, the mechanisms that drive the loss of SMA motoneurons remains poorly understood. Here, we describe an in vitro model of SMA-associated degeneration using primary motoneurons derived from Smn2B/- SMA mice and show that Fas activation selectively triggers death of the proximal motoneurons. Fas-induced death of SMA motoneurons has the molecular signature of the motoneuron-selective Fas death pathway that requires activation of p38 kinase, caspase-8, -9 and -3 as well as upregulation of collapsin response mediator protein 4 (CRMP4). In addition, Rho Kinase (ROCK) is required for Fas recruitment. Remarkably, we found that exogenous activation of Fas also promotes axonal elongation in both wildtype and SMA motoneurons. Axon outgrowth of motoneurons promoted by Fas requires the activity of ERK, ROCK and caspases. This work defines a dual role of Fas signaling in motoneurons that can elicit distinct responses from cell death to axonal growth.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene and low SMN protein levels. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy and clinical trials use a single intravenous bolus of the blood-brain-barrier penetrant scAAV9-cba-SMN by either systemic or central nervous system (CNS) delivery, resulting in impressive amelioration of the clinical phenotype but not a complete cure. The impact of scAAV9-cba-SMN treatment regimens on the CNS as well as on specific peripheral organs is yet to be described in a comparative manner. Therefore, we injected SMA mice with scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. In our system, ICV injections increased SMN in peripheral organs and the CNS while IV administration increased SMN in peripheral tissues only, largely omitting the CNS. Both treatments rescued several peripheral phenotypes while only ICV injections were neuroprotective. Surprisingly, both delivery routes resulted in a robust rescue effect on survival, weight, and motor function, which in IV-treated mice relied on peripheral SMN restoration but not on targeting the motor neurons. This demonstrates the independent contribution of peripheral organs to SMA pathology and suggests that treatments should not be restricted to motor neurons.

Skeletal muscle displays remarkable plasticity upon exercise and is also one of the organs most affected by aging. Despite robust evidence that aging is associated with loss of fast-twitch (type II) muscle fibers, the underlying mechanisms remain to be elucidated. Here, we identified an exercise-induced long noncoding RNA

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the SMN1 gene. Although lower motor neurons are a primary target, there is evidence that peripheral organ defects contribute to SMA. Current SMA gene therapy uses a single, high titre intravenous bolus of AAV9-SMN resulting in impressive, yet limited amelioration of the clinical phenotype. However, risks of this treatment include liver toxicity. Intrathecal administration is under clinical trial but was interrupted due to safety concerns in a concomitant animal study. As there is no direct comparison between the different delivery strategies while avoiding high dose toxicity, we injected SMA mice with low dose scAAV9-cba-SMN either intravenously (IV) for peripheral SMN restoration or intracerebroventricularly (ICV) for CNS-focused SMN restoration. Here, IV injections restored SMN in peripheral tissues but not CNS, while ICV injections mildly increased SMN in the periphery and the CNS. Consequently, only ICV treatment rescued motor neuron degeneration. Surprisingly, both treatments resulted in an impressive rescue of survival, weight, motor function, and peripheral phenotypes including liver and pancreas pathology. Our work highlights independent contributions of peripheral organs to SMA pathology and suggests that treatments should not be restricted to the motor neuron.Graphical Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease wherein motor neuron degeneration leads to muscle weakness, progressive paralysis, and death within 3–5 years of diagnosis. Currently, the cause of ALS is unknown but, as with several neurodegenerative diseases, the potential role of neuroinflammation has become an increasingly popular hypothesis in ALS research. Indeed, upregulation of neuroinflammatory factors have been observed in both ALS patients and animal models. One such factor is the inflammatory inducer NF-κB. Besides its connection to inflammation, NF-κB activity can be linked to several genes associated to familial forms of ALS, and many of the environmental risk factors of the disease stimulate NF-κB activation. Collectively, this has led many to hypothesize that NF-κB proteins may play a role in ALS pathogenesis. In this review, we discuss the genetic and environmental connections between NF-κB and ALS, as well as how this pathway may affect different CNS cell types, and finally how this may lead to motor neuron degeneration.

There is no FDA-approved medication for methamphetamine (METH) use disorder. New therapeutic approaches are needed, especially for people who use METH heavily and are at high risk for overdose. This study used genetically engineered rats to evaluate PARKIN as a potential target for METH use disorder. PARKIN knockout, PARKIN-overexpressing, and wild-type young adult male Long Evans rats were trained to self-administer high doses of METH using an extended-access METH self-administration paradigm. Reinforcing/rewarding properties of METH were assessed by quantifying drug-taking behavior and time spent in a METH-paired environment. PARKIN knockout rats self-administered more METH and spent more time in the METH-paired environment than wild-type rats. Wild-type rats overexpressing PARKIN self-administered less METH and spent less time in the METH-paired environment. PARKIN knockout rats overexpressing PARKIN self-administered less METH during the first half of drug self-administration days than PARKIN-deficient rats. The results indicate that rats with PARKIN excess or PARKIN deficit are useful models for studying neural substrates underlying “resilience” or vulnerability to METH use disorder and identify PARKIN as a novel potential drug target to treat heavy use of METH.

αSynuclein aggregation at the synapse is an early event in Parkinson’s disease and is associated with impaired striatal synaptic function and dopaminergic neuronal death. The cysteine string protein (CSPα) and αsynuclein have partially overlapping roles in maintaining synaptic function and mutations in each cause neurodegenerative diseases. CSPα is a member of the DNAJ/HSP40 family of co-chaperones and like αsynuclein, chaperones the SNARE complex assembly and neurotransmitter release. αSynuclein can rescue neurodegeneration in CSPαKO mice. However, whether αsynuclein aggregation alters CSPα expression and function is unknown. Here we show that αsynuclein aggregation at the synapse induces a decrease in synaptic CSPα and a reduction in the complexes that CSPα forms with HSC70 and STGa. We further show that viral delivery of CSPα rescues in vitro the impaired vesicle recycling in PC12 cells with αsynuclein aggregates and in vivo reduces synaptic αsynuclein aggregates restoring normal dopamine release in 1-120hαsyn mice. These novel findings reveal a mechanism by which αsynuclein aggregation alters CSPα at the synapse, and show that CSPα rescues αsynuclein aggregation-related phenotype in 1-120hαsyn mice similar to the effect of αsynuclein in CSPαKO mice. These results implicate CSPα as a potential therapeutic target for the treatment of early-stage PD.

International audience; Background: Parkinson's disease (PD) is characterized by a severe loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Perturbation of protein thiol redox homeostasis has been shown to play a role in the dysregulation of cell death and cell survival signaling pathways in these neurons. Glutaredoxin 1 (Grx1) is a thiol/disulfide oxidoreductase that catalyzes the deglutathionylation of proteins and is important for regulation of cellular protein thiol redox homeostasis.Objectives: We evaluated if the downregulation of Grx1 could lead to dopaminergic degeneration and PD-relevant motor deficits in mice.Methods: Grx1 was downregulated unilaterally through viral vector-mediated transduction of short hairpin RNA against Grx1 into the SNpc. Behavioral assessment was performed through rotarod and elevated body swing test. Stereological analysis of tyrosine hydroxylase-positive and Nissl-positive neurons was carried out to evaluate neurodegeneration.Results: Downregulation of Grx1 resulted in contralateral bias of elevated body swing and reduced latency to fall off, accelerating rotarod. This was accompanied by a loss of tyrosine hydroxylase-positive neurons in the SNpc and their DA projections in the striatum. Furthermore, there was a loss Nissl-positive neurons in the SNpc, indicating cell death. This was selective to the SNpc neurons because DA neurons in the ventral tegmental area were unaffected akin to that seen in human PD. Furthermore, Grx1 mRNA expression was substantially decreased in the SNpc from PD patients.Conclusions: Our study indicates that Grx1 is critical for the survival of SNpc DA neurons and that it is downregulated in human PD. © 2020 International Parkinson and Movement Disorder Society.

Background & aimsSpinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and death in children. SMA patients are more susceptible to dyslipidemia as well as liver steatosis, features reproduced in SMA mouse models. As current pre-clinical models of NAFLD are invariably imperfect and generally take a long time to develop, the rapid development of liver steatosis in SMA mice provides a means to identify molecular markers of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whetherSmn2B/-mice, a model of severe SMA, display typical features of NAFLD/non-alcoholic steatohepatitis (NASH).MethodsBiochemical, histological, electron microscopy, proteomic, and high-resolution respirometry were used.ResultsTheSmn2B/-mice develop steatohepatitis early in life. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in impaired hepatic function including alterations in protein output, complement, coagulation, iron homeostasis, and IGF-1 metabolism. The steatohepatitis is reversible by AAV9-SMN gene therapy. The NAFLD phenotype is likely due to non-esterified fatty acid (NEFA) overload from peripheral lipolysis, subsequent to hyperglucagonemia compounded by reduced muscle use. Mitochondrial β-oxidation contributed to hepatic damage as we observed enhanced hepatic mitochondrial β-oxidation and reactive oxygen species production. Hepatic mitochondrial content, however, was decreased. In contrast to typical NAFLD/NASH, theSmn2B/-mice lose weight due to their neurological condition, develop hypoglycemia and do not develop hepatic fibrosis.ConclusionTheSmn2B/-mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.

Spinal muscular atrophy (SMA) is a neuromuscular disorder affecting young children. While pre-clinical models of SMA show small spleens, the same is not true in humans. Here, we show by doppler ultrasonography decreased splenic blood flow in Smn2B/- mice. Further, AAV9-SMN gene therapy does not rescue the distal ear and tail necrosis nor the spleen size in these mice, suggesting that the latter may be linked to a cardiovascular defect. Absence of smaller spleens in human patients is likely due to differences in presentation of defects in SMA between pre-clinical mouse models and human patients, particularly the susceptibility to cardiovascular issues.

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells. Keywords: adeno-associated virus, AAV8, AAV9, suspension HEK293 cells, transient transfection, orbitally shaken bioreactors, immune affinity chromatography, CNS transduction

Summary Little information is available about how post-transcriptional mechanisms regulate the aging process. Here, we show that the RNA-binding protein Pumilio2 (PUM2), which is a translation repressor, is induced upon aging and acts as a negative regulator of lifespan and mitochondrial homeostasis. Multi-omics and cross-species analyses of PUM2 function show that it inhibits the translation of the mRNA encoding for the mitochondrial fission factor (Mff), thereby impairing mitochondrial fission and mitophagy. This mechanism is conserved in C. elegans by the PUM2 ortholog PUF-8. puf-8 knock-down in old nematodes and Pum2 CRISPR/Cas9-mediated knockout in the muscles of elderly mice enhances mitochondrial fission and mitophagy in both models, hence improving mitochondrial quality control and tissue homeostasis. Our data reveal how a PUM2-mediated layer of post-transcriptional regulation links altered Mff translation to mitochondrial dynamics and mitophagy, thereby mediating age-related mitochondrial dysfunctions.

Amyloid beta accumulation into insoluble plaques (Aβp) is known to play a significant role in the pathological process in Alzheimer's disease (AD). The presence of Aβp is also one of the neuropathological hallmarks for the disease. AD final diagnosis is generally acknowledged after the evaluation of Aβp deposition in the brain. Insoluble Aβp accumulation may also concur to cause AD as postulated in the so-called amyloid hypothesis. Therefore, the visualization, evaluation and quantification of Aβp are nowadays the keys for a better understanding of the disease, which may point to a possible cure for AD in the near future. Synchrotron-based X-ray phase contrast (XPC) has been demonstrated as the only imaging method that can retrieve the Aβp signal with high spatial resolution (up to 10 µm), high sensitivity and three-dimensional information at the same time. Although at the moment XPC is suitable for ex vivo samples only, it may develop into an alternative to positron emission tomography and magnetic resonance imaging in Aβp imaging. In this contribution the possibility of using synchrotron-based X-ray phase propagation computed tomography to visualize and measure Aβp on mouse brains is presented. A careful setup optimization for this application leads to a significant improvement of spatial resolution (∼1 µm), data acquisition speed (five times faster), X-ray dose (five times lower) and setup complexity, without a substantial loss in sensitivity when compared with the classic implementation of grating-based X-ray interferometry., Journal of Synchrotron Radiation, 23 (3), ISSN:0909-0495, ISSN:1600-5775

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.

Severe spinal cord contusions interrupt nearly all brain projections to lumbar circuits producing leg movement. Failure of these projections to reorganize leads to permanent paralysis. Here we modeled these injuries in rodents. A severe contusion abolished all motor cortex projections below injury. However, the motor cortex immediately regained adaptive control over the paralyzed legs during electrochemical neuromodulation of lumbar circuits. Glutamatergic reticulospinal neurons with residual projections below the injury relayed the cortical command downstream. Gravity-assisted rehabilitation enabled by the neuromodulation therapy reinforced these reticulospinal projections, rerouting cortical information through this pathway. This circuit reorganization mediated a motor cortex-dependent recovery of natural walking and swimming without requiring neuromodulation. Cortico-reticulo-spinal circuit reorganization may also improve recovery in humans.

6 páginas, 3 figuras. En material suplementario 3 tablas, 7 figuras., The chances to develop Alzheimer’s disease (AD) result from a combination of genetic and non-genetic risk factors1, the latter likely mediated by epigenetic mechanisms2. In the past, genome-wide association studies (GWAS) have identified an important number of risk loci associated with AD pathology3, but a causal relationship thereof remains difficult to establish. In contrast, locus-specific or epigenome-wide association studies (EWAS) have revealed site-specific epigenetic alterations and thereby provide mechanistic insights for a particular risk gene, but often lack the statistical power of GWAS4. Here, combining both approaches, we report a hitherto unidentified association of the Peptidase M20 domain-containing protein 1 (PM20D1) with AD. We find that PM20D1 is a methylation/expression quantitative trait locus (mQTL/eQTL) coupled to an AD-risk associated haplotype, which displays enhancer-like characteristics and contacts the PM20D1 promoter via a haplotype-dependent, CTCF-mediated chromatin loop. Furthermore, PM20D1 is increased following AD-related neurotoxic insults, at symptomatic stages in the APP/PS1 mouse model of AD and in human AD patients, who are carriers of the non-risk haplotype. Importantly, genetically increasing and decreasing the expression of PM20D1 reduces and aggravates AD-related pathologies, respectively. These findings suggest that in a particular genetic background, PM20D1 contributes to neuroprotection against AD., The work in the laboratory of J.G. is supported by the SYNAPSIS Foundation, the Béatrice Ederer-Weber Stiftung, the Floshield Foundation and the Alzheimer’s Association (grant no. NIRG-15-363964). The laboratory of D.M. is supported by the Foundation Jérôme Lejeune, Spanish Ministerio de Educación y Competitividad (grant no. BFU2014-53093). The laboratory of J.P.-T. is supported by the Spanish Ministerio de Economía, Industria y Competitividad and the FEDER programme from the EU (grant no. SAF2014-59469-R) and the CIBERNED. J.V.S.-M. is supported by a SYNAPSIS Foundation Fellowship for Advanced PostDocs and the Heidi Seiler-Stiftung foundation. H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII). B.A.S. is an EMBO long-term fellow (ALTF 1605-2014, Marie Curie Actions, LTFCOFUND2013, GA-2013-609409). A.M.-S. is a recipient of a FPI PhD studentship from MINECO. M.E. is an ICREA Research Professor. J.G. is an MQ fellow and a NARSAD Independent Investigator.

Here we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium in orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI) mediated transfection of a 2-plasmid system and is specified for production in milliliter to liter scales. After PEI and plasmid DNA (pDNA) complex formation the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 3-day batch process, cell cultures are further processed using different methods for lysis and recovery. Methods for the purification of viral particles are described, including iodixanol gradient purification, immunoaffinity chromatography, and ultrafiltration, as well as quantitative PCR to quantify vector titer.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 contains functional GTPase and kinase domains. The most common G2019S mutation enhances the kinase activity of LRRK2 in vitro whereas G2019S LRRK2 expression in cultured neurons induces toxicity in a kinase-dependent manner. These observations suggest a potential role for kinase activity in LRRK2-associated PD. We have recently developed a novel rodent model of PD with progressive neurodegeneration induced by the adenoviral-mediated expression of G2019S LRRK2. In the present study, we further characterize this LRRK2 model and determine the contribution of kinase activity to LRRK2-mediated neurodegeneration. Recombinant human adenoviral vectors were employed to deliver human wild-type, G2019S or kinase-inactive G2019S/D1994N LRRK2 to the rat striatum. LRRK2-dependent pathology was assessed in the striatum, a region where LRRK2 protein is normally enriched in the mammalian brain. Human LRRK2 variants are robustly expressed throughout the rat striatum. Expression of G2019S LRRK2 selectively induces the accumulation of neuronal ubiquitin-positive inclusions accompanied by neurite degeneration and the altered distribution of axonal phosphorylated neurofilaments. Importantly, the introduction of a kinase-inactive mutation (G2019S/D1994N) completely ameliorates the pathological effects of G2019S LRRK2 in the striatum supporting a kinase activity-dependent mechanism for this PD-associated mutation. Collectively, our study further elucidates the pathological effects of the G2019S mutation in the mammalian brain and supports the development of kinase inhibitors as a potential therapeutic approach for treating LRRK2-associated PD. This adenoviral rodent model provides an important tool for elucidating the molecular basis of LRRK2-mediated neurodegeneration.

Severe spinal cord contusions interrupt nearly all brain projections to lumbar circuits producing leg movement. Failure of these projections to reorganize leads to permanent paralysis. Here we modeled these injuries in rodents. A severe contusion abolished all motor cortex projections below injury. However, the motor cortex immediately regained adaptive control over the paralyzed legs during electrochemical neuromodulation of lumbar circuits. Glutamatergic reticulospinal neurons with residual projections below the injury relayed the cortical command downstream. Gravity-assisted rehabilitation enabled by the neuromodulation therapy reinforced these reticulospinal projections, rerouting cortical information through this pathway. This circuit reorganization mediated a motor cortex-dependent recovery of natural walking and swimming without requiring neuromodulation. Cortico-reticulo-spinal circuit reorganization may also improve recovery in humans.

Tau pathology is associated with cognitive decline in Alzheimer’s disease, and missense tau mutations cause frontotemporal dementia. Hyperphosphorylation and misfolding of tau are considered critical steps leading to tauopathies. Here, we determine how motifs controlling conformational changes in the microtubule-binding domain determine tau pathology in vivo. Human tau was overexpressed in the adult mouse forebrain to compare variants carrying residues that modulate tau propensity to acquire a β-sheet conformation. The P301S mutation linked to frontotemporal dementia causes tau aggregation and rapidly progressing motor deficits. By comparison, wild-type tau becomes heavily hyperphosphorylated, and induces behavioral impairments that do not progress over time. However, the behavioral defects caused by wild-type tau can be suppressed when β-sheet breaking proline residues are introduced in the microtubule-binding domain of tau. This modification facilitates tau interaction with microtubules, as shown by lower levels of phosphorylation, and by the enhanced protective effects of mutated tau against the severing of the cytoskeleton in neurons exposed to vinblastine. Altogether, motifs that are critical for tau conformation determine interaction with microtubules and subsequent pathological modifications, including phosphorylation and aggregation.

The deposition of amyloid-beta (Aβ) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aβ is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aβ production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aβ generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aβ. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aβ production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aβ production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.

Microphthalmia-associated transcription factor (MITF) is a master regulator of pigmented cell survival and differentiation with direct transcriptional links to cell cycle, apoptosis and pigmentation. In mouse, Mitf is expressed early and uniformly in optic vesicle (OV) cells as they evaginate from the developing neural tube, and null Mitf mutations result in microphthalmia and pigmentation defects. However, homozygous mutations in MITF have not been identified in humans; therefore, little is known about its role in human retinogenesis. We used a human embryonic stem cell (hESC) model that recapitulates numerous aspects of retinal development, including OV specification and formation of retinal pigment epithelium (RPE) and neural retina progenitor cells (NRPCs), to investigate the earliest roles of MITF. During hESC differentiation toward a retinal lineage, a subset of MITF isoforms was expressed in a sequence and tissue distribution similar to that observed in mice. In addition, we found that promoters for the MITF-A, -D and -H isoforms were directly targeted by Visual Systems Homeobox 2 (VSX2), a transcription factor involved in patterning the OV toward a NRPC fate. We then manipulated MITF RNA and protein levels at early developmental stages and observed decreased expression of eye field transcription factors, reduced early OV cell proliferation and disrupted RPE maturation. This work provides a foundation for investigating MITF and other highly complex, multi-purposed transcription factors in a dynamic human developmental model system.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). LRRK2 mutations typically give rise to Lewy pathology in the brains of PD subjects yet can induce tau-positive neuropathology in some cases. The pathological interaction between LRRK2 and tau remains poorly defined. To explore this interaction in vivo, we crossed a well-characterized human P301S-tau transgenic mouse model of tauopathy with human G2019S-LRRK2 transgenic mice or LRRK2 knockout (KO) mice. We find that endogenous or pathogenic LRRK2 expression has minimal effects on the steady-state levels, solubility and abnormal phosphorylation of human P301S-tau throughout the mouse brain. We next developed a new model of tauopathy by delivering AAV2/6 vectors expressing human P301S-tau to the hippocampal CA1 region of G2019S-LRRK2 transgenic or LRRK2 KO mice. P301S-tau expression induces hippocampal tau pathology and marked degeneration of CA1 pyramidal neurons in mice, however, this occurs independently of endogenous or pathogenic LRRK2 expression. We further developed new AAV2/6 vectors co-expressing human WT-tau and GFP to monitor the neuron-to-neuron transmission of tau within defined hippocampal neuronal circuits. While endogenous LRRK2 is not required for tau transmission, we find that G2019S-LRRK2 markedly enhances the neuron-to-neuron transmission of tau in mice. Our data suggest that mutant tau-induced neuropathology occurs independently of LRRK2 expression in two mouse models of tauopathy but identifies a novel pathogenic role for G2019S-LRRK2 in promoting the neuronal transmission of WT-tau protein. These findings may have important implications for understanding the development of tau neuropathology in LRRK2-linked PD brains.

Recent progress in the understanding of neurodegenerative diseases revealed that multiple molecular mechanisms contribute to pathological changes in neurons. A large fraction of these alterations can be linked to dysfunction in the endoplasmic reticulum (ER) and mitochondria, affecting metabolism and secretion of lipids and proteins, calcium homeostasis, and energy production. Remarkably, these organelles are interacting with each other at specialized domains on the ER called mitochondria-associated membranes (MAMs). These membrane structures rely on the interaction of several complexes of proteins localized either at the mitochondria or at the ER interface and serve as an exchange platform of calcium, metabolites, and lipids, which are critical for the function of both organelles. In addition, recent evidence indicates that MAMs also play a role in the control of mitochondria dynamics and autophagy. MAMs thus start to emerge as a key element connecting many changes observed in neurodegenerative diseases. This review will focus on the role of MAMs in amyotrophic lateral sclerosis (ALS) and hereditary motor and sensory neuropathy, two neurodegenerative diseases particularly affecting neurons with long projecting axons. We will discuss how defects in MAM signaling may impair neuronal calcium homeostasis, mitochondrial dynamics, ER function, and autophagy, leading eventually to axonal degeneration. The possible impact of MAM dysfunction in glial cells, which may affect the capacity to support neurons and/or axons, will also be described. Finally, the possible role of MAMs as an interesting target for development of therapeutic interventions aiming at delaying or preventing neurodegeneration will be highlighted.

Genetically encoded calcium indicators (GECIs) enable imaging of in vivo brain cell activity with high sensitivity and specificity. In contrast to viral infection or in utero electroporation, indicator expression in transgenic reporter lines is induced noninvasively, reliably, and homogenously. Recently, Cre/tTA-dependent reporter mice were introduced, which provide high-level expression of green fluorescent GECIs in a cell-type-specific and inducible manner when crossed with Cre and tTA driver mice. Here, we generated and characterized the first red-shifted GECI reporter line of this type using R-CaMP1.07, a red fluorescent indicator that is efficiently two-photon excited above 1000 nm. By crossing the new R-CaMP1.07 reporter line to Cre lines driving layer-specific expression in neocortex we demonstrate its high fidelity for reporting action potential firing in vivo, long-term stability over months, and versatile use for functional imaging of excitatory neurons across all cortical layers, especially in the previously difficult to access layers 4 and 6. ISSN:1932-6203

Direct collection of extracellular fluid (ECF) plays a central role in the monitoring of neurological disorders. Current approaches using microdialysis catheters are however drastically limited in term of temporal resolution. Here we show a functional in vivo validation of a droplet collection system included at the tip of a neural probe. The system comprises an advanced droplet formation mechanism which enables the collection of neurochemicals present in the brain ECF at high-temporal resolution. The probe was implanted in a rat brain and could successfully collect fluid samples organized in a train of droplets. A microfabricated target plate compatible with most of the surface-based detection methods was specifically developed for sample analysis. The time-resolved brain-fluid samples are analyzed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results provide a time evolution picture of the cerebral tissues neurochemical composition for selected elements known for their involvement in neurodegenerative diseases., Current approaches studying the collection of extracellular fluid to monitor neurological disorders have temporal resolution limitations. Here the authors show functional in vivo validation of a droplet collection system included at the tip of a neural probe.

Reference EPFL-ARTICLE-227589doi:10.1051/medsci/20173301013View record in Web of Science Record created on 2017-05-01, modified on 2017-05-01

Parkinson's disease is a neurodegenerative disorder characterized by the loss of neurons in specific regions of the nervous system, notably in the substantia nigra pars compacta and, in most cases, by the deposition of intraneuronal inclusions named Lewy bodies. These pathological alterations have profound effects on the brain function, leading to the progressive development of various symptoms, the most prominent being the impaired initiation of voluntary movements caused by the loss of dopamine signaling in the basal ganglia. Here, we provide an overview of the mouse models of Parkinson's disease, with the goal of guiding selection of the most appropriate model for studying the question at hand. Pharmacological approaches targeting dopamine signaling and toxins leading to selective degeneration of nigral neurons are used to validate symptomatic treatments that aim at restoring effective dopaminergic function for motor control. Alternative mouse models are based on genetic modifications that are meant to reproduce the inherited alterations associated with familial forms of Parkinson's disease. Although genetic models have most often failed to induce overt degeneration of nigral dopaminergic neurons, they provide essential tools to explore the multifactorial etiology of this complex neurodegenerative disorder. Curr. Protoc. Mouse Biol. 4:121-139 © 2014 by John Wiley & Sons, Inc. Keywords: Parkinson's disease; mouse models of neurodegeneration; genetic factors; toxins

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatmentin vitroinduced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.

Parkinson's disease (PD) is characterized by the selective degeneration of neuronal populations presumably due to pathogenic interactions between aging and predisposing factors such as increased levels of a-synuclein. Here, we genetically modulate the activity of the transcription factor Forkhead box protein O-3 (FOXO3) in adult nigral dopaminergic neurons using viral vectors and explore how this determinant of longevity impacts on neuronal fate in normal and diseased conditions. We find that dopaminergic neurons are particularly vulnerable to changes in FOXO3 activity in the substantia nigra. While constitutive activation has proapoptotic effects leading to neuronal loss, inhibition of FOXO-mediated transcription by a dominant-negative competitor causes oxidative damage and is detrimental at high vector dose. To address the role of FOXO3 in PD, we modulate its activity in dopaminergic neurons overexpressing human a-synuclein. In this pathogenic condition, we find that FOXO inhibition has protective effects, suggesting that this transcription factor ultimately contributes to neuronal cell death. Nevertheless, mild FOXO3 activity also protects nigral neurons against the accumulation of human a-synuclein, albeit to a lesser extent. FOXO3 reduces the amount of a-synuclein present in the soluble protein fraction and promotes the coalescence of dense proteinase K-resistant aggregates, with an accumulation of autophagic vacuoles containing lipofuscin. Consistent with these in vivo observations, we find that FOXO3 controls autophagic flux in neuronal cells. Altogether, these results point to FOXO3 as an important determinant of neuronal survival in the substantia nigra, which may oppose a-synuclein accumulation and proteotoxicity.

With the development of effective systems for gene delivery to the central nervous system (CNS), gene therapy has become a therapeutic option for the treatment of Parkinson's disease (PD). Gene therapies that are the most advanced in the clinic have been designed to more effectively compensate for the lack of dopamine signaling in the basal ganglia and rescue the cardinal motor symptoms of PD. However, it remains essential to devise novel therapies to prevent neurodegeneration and disease progression. Since gene therapy has been initially proposed for the delivery of neurotrophins to support the survival and function of dopaminergic neurons, our understanding of PD etiology has changed dramatically. Genes implicated in familial forms of the disease and genetic risk factors associated with sporadic PD have been identified. The spreading of the α-synuclein pathology, as well as perturbations of the lysosomal and mitochondrial activities, appear to play critical roles in the pathogenesis. These findings provide novel targets for gene therapy against PD, but at the same time underline the complexity of this chronic disease. Here we review and discuss the successes and limitations of gene therapy approaches, which have been proposed to provide neuroprotection in PD.