Single-cell and spatial atlases of spinal cord injury in the Tabulae Paralytica.

Here, we introduce the Tabulae Paralytica-a compilation of four atlases of spinal cord injury (SCI) comprising a single-nucleus transcriptome atlas of half a million cells, a multiome atlas pairing transcriptomic and epigenomic measurements within the same nuclei, and two spatial transcriptomic atlases of the injured spinal cord spanning four spatial and temporal dimensions. We integrated these atlases into a common framework to dissect the molecular logic that governs the responses to injury within the spinal cord1. The Tabulae Paralytica uncovered new biological principles that dictate the consequences of SCI, including conserved and divergent neuronal responses to injury; the priming of specific neuronal subpopulations to upregulate circuit-reorganizing programs after injury; an inverse relationship between neuronal stress responses and the activation of circuit reorganization programs; the necessity of re-establishing a tripartite neuroprotective barrier between immune-privileged and extra-neural environments after SCI and a failure to form this barrier in old mice. We leveraged the Tabulae Paralytica to develop a rejuvenative gene therapy that re-established this tripartite barrier, and restored the natural recovery of walking after paralysis in old mice. The Tabulae Paralytica provides a window into the pathobiology of SCI, while establishing a framework for integrating multimodal, genome-scale measurements in four dimensions to study biology and medicine.

The RNA-Binding Protein PUM2 Impairs Mitochondrial Dynamics and Mitophagy During Aging.

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.

Long term peripheral AAV9-SMN gene therapy promotes survival in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is caused by the loss of the SMN1 gene and low SMN protein levels. Current SMA therapies work by increasing SMN protein in the body. Although SMA is regarded as a motor neuron disorder, growing evidence shows that several peripheral organs contribute to SMA pathology. A gene therapy treatment, onasemnogene abeparvovec, is being explored in clinical trials via both systemic and central nervous system (CNS) specific delivery, but the ideal route of delivery as well as the long-term effectiveness is unclear. To investigate the impact of gene therapy long term, we assessed SMA mice at 6 months after treatment of either intravenous (IV) or intracerebroventricular (ICV) delivery of scAAV9-cba-SMN. Interestingly, we observed that SMN protein levels were restored in the peripheral tissues but not in the spinal cord at 6 months of age. However, ICV injections provided better motor neuron and motor function protection than IV injection, while IV-injected mice demonstrated better protection of neuromuscular junctions and muscle fiber size. Surprisingly, both delivery routes resulted in an equal rescue on survival, weight, and liver and pancreatic defects. These results demonstrate that continued peripheral AAV9-SMN gene therapy is beneficial for disease improvement even in the absence of SMN restoration in the spinal cord.

AAV9-mediated SMN gene therapy rescues cardiac desmin but not lamin A/C and elastin dysregulation in Smn2B/- spinal muscular atrophy mice.

Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/- SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/- mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/- mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.

Differential effect of Fas activation on spinal muscular atrophy motoneuron death and induction of axonal growth.

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 indicates 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-associated 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.

Central and peripheral delivered AAV9-SMN are both efficient but target different pathomechanisms in a mouse model of spinal muscular atrophy.

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.

Astrocyte-targeting RNA interference against mutated superoxide dismutase 1 induces motoneuron plasticity and protects fast-fatigable motor units in a mouse model of amyotrophic lateral sclerosis.

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 SOD1G93A protein. An AAV-gfaABC1 D vector encoding an artificial microRNA is used to deliver RNA interference against mutated SOD1 selectively in astrocytes. The treatment leads to the progressive rescue of neuromuscular junction occupancy, to the recovery of the compound muscle action potential in the gastrocnemius muscle, and significantly improves neuromuscular function. In the spinal cord, gene therapy targeting astrocytes protects a small pool of the most vulnerable fast-fatigable MN until disease end stage. In the gastrocnemius muscle of the treated SOD1G93A mice, the fast-twitch type IIB muscle fibers are preserved from atrophy. Axon collateral sprouting is observed together with muscle fiber type grouping indicative of denervation/reinnervation events. The transcriptome profiling of spinal cord MN shows changes in the expression levels of factors regulating the dynamics of microtubules. Gene therapy delivering RNA interference against mutated SOD1 in astrocytes protects fast-fatigable motor units and thereby improves neuromuscular function in ALS mice.

CSPα reduces aggregates and rescues striatal dopamine release in α-synuclein transgenic mice.

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.

Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice.

AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.

Altered interplay between endoplasmic reticulum and mitochondria in Charcot-Marie-Tooth type 2A neuropathy.

Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.

The Links between ALS and NF-κB.

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.

Dopamine and Methamphetamine Differentially Affect Electron Transport Chain Complexes and Parkin in Rat Striatum: New Insight into Methamphetamine Neurotoxicity.

Methamphetamine (METH) is a highly abused psychostimulant that is neurotoxic to dopaminergic (DAergic) nerve terminals in the striatum and increases the risk of developing Parkinson's disease (PD). In vivo, METH-mediated DA release, followed by DA-mediated oxidative stress and mitochondrial dysfunction in pre- and postsynaptic neurons, mediates METH neurotoxicity. METH-triggered oxidative stress damages parkin, a neuroprotective protein involved in PD etiology via its involvement in the maintenance of mitochondria. It is not known whether METH itself contributes to mitochondrial dysfunction and whether parkin regulates complex I, an enzymatic complex downregulated in PD. To determine this, we separately assessed the effects of METH or DA alone on electron transport chain (ETC) complexes and the protein parkin in isolated striatal mitochondria. We show that METH decreases the levels of selected complex I, II, and III subunits (NDUFS3, SDHA, and UQCRC2, respectively), whereas DA decreases the levels only of the NDUFS3 subunit in our preparations. We also show that the selected subunits are not decreased in synaptosomal mitochondria under similar experimental conditions. Finally, we found that parkin overexpression does not influence the levels of the NDUFS3 subunit in rat striatum. The presented results indicate that METH itself is a factor promoting dysfunction of striatal mitochondria; therefore, it is a potential drug target against METH neurotoxicity. The observed decreases in ETC complex subunits suggest that DA and METH decrease activities of the ETC complexes via oxidative damage to their subunits and that synaptosomal mitochondria may be somewhat "resistant" to DA- and METH-induced disruption in mitochondrial ETC complexes than perikaryal mitochondria. The results also suggest that parkin does not regulate NDUFS3 turnover in rat striatum.

G2019S LRRK2 enhances the neuronal transmission of tau in the mouse brain.

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.

Glutaredoxin 1 Downregulation in the Substantia Nigra Leads to Dopaminergic Degeneration in Mice.

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.

Parkin functionally interacts with PGC-1α to preserve mitochondria and protect dopaminergic neurons.

To understand the cause of Parkinson's disease (PD), it is important to determine the functional interactions between factors linked to the disease. Parkin is associated with autosomal recessive early-onset PD, and controls the transcription of PGC-1α, a master regulator of mitochondrial biogenesis. These two factors functionally interact to regulate the turnover and quality of mitochondria, by increasing both mitophagic activity and mitochondria biogenesis. In cortical neurons, co-expressing PGC-1α and Parkin increases the number of mitochondria, enhances maximal respiration, and accelerates the recovery of the mitochondrial membrane potential following mitochondrial uncoupling. PGC-1α enhances Mfn2 transcription, but also leads to increased degradation of the Mfn2 protein, a key ubiquitylation target of Parkin on mitochondria. In vivo, Parkin has significant protective effects on the survival and function of nigral dopaminergic neurons in which the chronic expression of PGC-1α is induced. Ultrastructural analysis shows that these two factors together control the density of mitochondria and their interaction with the endoplasmic reticulum. These results highlight the combined effects of Parkin and PGC-1α in the maintenance of mitochondrial homeostasis in dopaminergic neurons. These two factors synergistically control the quality and function of mitochondria, which is important for the survival of neurons in Parkinson's disease.

Behaviour-dependent recruitment of long-range projection neurons in somatosensory cortex.

In the mammalian neocortex, segregated processing streams are thought to be important for forming sensory representations of the environment, but how local information in primary sensory cortex is transmitted to other distant cortical areas during behaviour is unclear. Here we show task-dependent activation of distinct, largely non-overlapping long-range projection neurons in the whisker region of primary somatosensory cortex (S1) in awake, behaving mice. Using two-photon calcium imaging, we monitored neuronal activity in anatomically identified S1 neurons projecting to secondary somatosensory (S2) or primary motor (M1) cortex in mice using their whiskers to perform a texture-discrimination task or a task that required them to detect the presence of an object at a certain location. Whisking-related cells were found among S2-projecting (S2P) but not M1-projecting (M1P) neurons. A higher fraction of S2P than M1P neurons showed touch-related responses during texture discrimination, whereas a higher fraction of M1P than S2P neurons showed touch-related responses during the detection task. In both tasks, S2P and M1P neurons could discriminate similarly between trials producing different behavioural decisions. However, in trials producing the same decision, S2P neurons performed better at discriminating texture, whereas M1P neurons were better at discriminating location. Sensory stimulus features alone were not sufficient to elicit these differences, suggesting that selective transmission of S1 information to S2 and M1 is driven by behaviour.

PFN2 and GAMT as common molecular determinants of axonal Charcot-Marie-Tooth disease.

BACKGROUND: Charcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes. METHODS: A proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models. RESULTS: Proteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves. CONCLUSIONS: We unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.

Alpha-synuclein ferrireductase activity is detectible in vivo, is altered in Parkinson's disease and increases the neurotoxicity of DOPAL.

The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease.

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aß production in the brain.

The deposition of amyloid-beta (Aß) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aß 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 Aß 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 Aß 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 Aß. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aß production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aß 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.

A subcutaneous cellular implant for passive immunization against amyloid-ß reduces brain amyloid and tau pathologies.

Passive immunization against misfolded toxic proteins is a promising approach to treat neurodegenerative disorders. For effective immunotherapy against Alzheimer's disease, recent clinical data indicate that monoclonal antibodies directed against the amyloid-ß peptide should be administered before the onset of symptoms associated with irreversible brain damage. It is therefore critical to develop technologies for continuous antibody delivery applicable to disease prevention. Here, we addressed this question using a bioactive cellular implant to deliver recombinant anti-amyloid-ß antibodies in the subcutaneous tissue. An encapsulating device permeable to macromolecules supports the long-term survival of myogenic cells over more than 10 months in immunocompetent allogeneic recipients. The encapsulated cells are genetically engineered to secrete high levels of anti-amyloid-ß antibodies. Peripheral implantation leads to continuous antibody delivery to reach plasma levels that exceed 50 µg/ml. In a proof-of-concept study, we show that the recombinant antibodies produced by this system penetrate the brain and bind amyloid plaques in two mouse models of the Alzheimer's pathology. When encapsulated cells are implanted before the onset of amyloid plaque deposition in TauPS2APP mice, chronic exposure to anti-amyloid-ß antibodies dramatically reduces amyloid-ß40 and amyloid-ß42 levels in the brain, decreases amyloid plaque burden, and most notably, prevents phospho-tau pathology in the hippocampus. These results support the use of encapsulated cell implants for passive immunotherapy against the misfolded proteins, which accumulate in Alzheimer's disease and other neurodegenerative disorders.