Cysteine String Protein Alpha Research Articles

Assessing the Co-Chaperone and Synaptic Protein DNAJC5 as a Therapeutic Target in Alzheimer’s Disease (P1-6.010)

<h3>Objective:</h3> Assess the therapeutic potential of <i>DNAJC5</i> in Alzheimer’s disease (AD). <h3>Background:</h3> <i>DNAJC5</i> encodes cysteine-string protein alpha (CSPα), whose mutations cause adult neuronal ceroid lipofuscinosis (ANCL). ANCL is characterized by endolysosomal dysfunction, synaptic loss, and protein aggregate accumulation, similar to AD. CSPα facilitates secretion of the neurodegenerative proteins tau, TDP-43, and α-synuclein and maintains synaptic integrity. Therefore, CSPα is a promising therapeutic target. <h3>Design/Methods:</h3> N2A695 cells stably expressing mutant <i>DNAJC5</i> or knocking down endogenous <i>Dnajc5</i> were analyzed through immunoblotting and ELISA for APP processing and Aβ production. Brains of ANCL patients and <i>Dnajc5</i>+/−; 5xFAD mice were assessed for amyloid pathology with immunohistochemistry. Transcriptomics from ANCL, AD, and control brains was analyzed for differentially expressed genes (DEGs). The Clue.IO database was used to identify potential targets for gene therapy or pharmacological intervention. <h3>Results:</h3> ANCL-patient brains exhibited low soluble or insoluble Aβ levels, but intra-neuronal Aβ accumulation. They exhibited reductions in synaptic protein levels (SNAP-25, Synaptobrevin, Syntaxin 1, and Synaptophysin). Transcriptomic analysis of ANCL-patient brains showed reduced <i>TSPAN14</i>, which facilitates ADAM10 maturation, elevated <i>APH1B</i>, a γ-secretase subunit, and elevated <i>GSAP</i>, the γ-secretase activating protein. There were 67 upregulated and 15 downregulated transcripts associated with processes essential for synaptic function, including the MAPK cascade (<i>FYN</i>, <i>SOS1</i>), dendritic spine organization (<i>FYN</i>), and endosomal vesicle fusion (<i>VPS11</i>, <i>VPS39</i>). Clue.IO identified heat-shock protein inhibitors and an opioid receptor antagonist (BNTX) as potential pharmacological agents to counteract the effects of mutant <i>DNAJC5</i>. AD patients and 5xFAD mice exhibited reduced <i>DNAJC5</i> expression. Preliminary results from <i>Dnajc5</i>+/−; 5xFAD mice showed increased plaques in the corpus collosum and hippocampus. N2A695 cells expressing <i>DNAJC5</i> mutants showed increased Aβ while <i>DNAJC5</i> knockdown decreased Aβ. <h3>Conclusions:</h3> Low levels of CSPα alter Ab secretion and dysregulate genes and synaptic proteins, suggesting that increasing CSPα levels could reduce Aβ and improve synaptic function. <b>Disclosure:</b> Dr. Rosene has nothing to disclose. Dr. Nykanen has nothing to disclose. Kevin O’Dell has nothing to disclose. Ms. Nunez has nothing to disclose. Dr. Harari has nothing to disclose. John Cirrito has nothing to disclose. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for M6P Therapeutics. Prof. Sands has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Amicus. Prof. Sands has stock in JAYA Biosciences. The institution of Prof. Sands has received research support from JAYA Biosciences. Prof. Sands has received intellectual property interests from a discovery or technology relating to health care. Bruno Benitez has nothing to disclose.

Cysteine string protein alpha accumulates with early pre-synaptic dysfunction in Alzheimer's disease.

In Alzheimer’s disease, synapse loss causes memory and cognitive impairment. However, the mechanisms underlying synaptic degeneration in Alzheimer’s disease are not well understood. In the hippocampus, alterations in the level of cysteine string protein alpha, a molecular co-chaperone at the pre-synaptic terminal, occur prior to reductions in synaptophysin, suggesting that it is a very sensitive marker of synapse degeneration in Alzheimer’s. Here, we identify putative extracellular accumulations of cysteine string alpha protein, which are proximal to beta-amyloid deposits in post-mortem human Alzheimer’s brain and in the brain of a transgenic mouse model of Alzheimer’s disease. Cysteine string protein alpha, at least some of which is phosphorylated at serine 10, accumulates near the core of beta-amyloid deposits and does not co-localize with hyperphosphorylated tau, dystrophic neurites or glial cells. Using super-resolution microscopy and array tomography, cysteine string protein alpha was found to accumulate to a greater extent than other pre-synaptic proteins and at a comparatively great distance from the plaque core. This indicates that cysteine string protein alpha is most sensitive to being released from pre-synapses at low concentrations of beta-amyloid oligomers. Cysteine string protein alpha accumulations were also evident in other neurodegenerative diseases, including some fronto-temporal lobar dementias and Lewy body diseases, but only in the presence of amyloid plaques. Our findings are consistent with suggestions that pre-synapses are affected early in Alzheimer’s disease, and they demonstrate that cysteine string protein alpha is a more sensitive marker for early pre-synaptic dysfunction than traditional synaptic markers. We suggest that cysteine string protein alpha should be used as a pathological marker for early synaptic disruption caused by beta-amyloid.

DNAJC5 affects the endo‐lysosomal pathway, APP processing, and AD pathology in vitro and in vivo

The DnaJ heat shock protein family member C5 (DNAJC5) gene encodes Cysteine String Protein-alpha (CSPα). CSPα is a key endo-lysosomal element of the misfolding-associated protein secretion (MAPS) machinery. MAPS eliminates misfolded cytosolic proteins, including alpha-synuclein, tau, TDP-43, huntingtin. Mutations in the DNAJC5 gene cause rare early-onset dementia called adult-onset Neuronal ceroid lipofuscinosis (ANCL). Data from CSPα-deficient mice and flies suggest that CSPα is critical for preventing age-dependent neurodegeneration. The endo-lysosome plays an essential role in normal and abnormal Amyloid-beta precursor protein (APP) processing and subsequent β-amyloidogenesis in Alzheimer's disease (AD). However, the role of CSPα in APP processing, trafficking, and amyloidogenesis is not well understood.We used histological staining and whole transcriptome data from ANCL, AD patients, and age-matched pathology-free controls. Differential expression (DE) analysis was performed using DESeq2 software. We performed histological analysis of 5XFAD mouse models crossed with DNAJC5 mice. We used mouse neuroblastoma (N2A) cells stably expressing wild-type human APP695 (N2A695) and human wild-type (WT) and mutant DNAJC5. We used ELISA to quantify Aβ40 and Aβ42 in cell culture media and human brain lysates.CSPα co-localizes with endo-lysosomal and synaptic markers in N2A695 cells. CSPα overexpression affects lysosomal function and SNAP29-mediated exocytosis. Overexpression and knockdown of hCSPα-WT in N2A695 cells significantly affect extracellular Aβ40, Aβ42, full-length APP, and APP C-terminal fragments (CTF). N2A-APP cells expressing a gain-of-function DNAJC5 mutant displayed a significant increase in lysosomal and autophagy (LC3-II and p62) proteins, lysosomal exocytosis, and secreted levels of Aβ40 and Aβ42. ANCL brains showed considerable neuronal Aβ accumulation. ANCL brains exhibit a significant reduction of soluble and insoluble Aβ4 and Aβ42. Transcriptome analysis from ANCL brains shows changes in the mTOR pathway. DNAJC5 transcript levels are significantly reduced in AD cases compared to controls. A mouse AD model exhibits an inverse correlation between DNAJC5 transcript levels and Aβ plaques. 5XFAD mice haploinsufficient for DNAJC5 gene significantly increased the Aβ plaque burden and decreased Aβ plaque latency.Our results provide evidence of the novel and unexpected role of CSPα in endo-lysosomal function, lysosomal exocytosis, β-amyloidogenesis both in vitro and in vivo.

Autosomal-dominant adult neuronal ceroid lipofuscinosis caused by duplication in DNAJC5 initially missed by Sanger and whole-exome sequencing.

Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.

Therapeutic Effect of a Novel Fatty Acid Amide Hydrolase Inhibitor PF04457845 in the Repetitive Closed Head Injury Mouse Model.

Concussive traumatic brain injury (TBI) is the predominant type of brain injury in young adults and is a risk factor for the development of chronic traumatic encephalopathy and other neurodegenerative diseases late in life. Using a repetitive closed head injury mouse model, we found that treatment with PF04457845, a novel fatty acid amide hydrolase (FAAH) inhibitor that selectively elevated the brain levels of anandamide, improved locomotor function, learning, and memory in TBI mice examined by beam walk, Y-maze, and Morris water maze tests. The accumulation of microglia and astrocytes and the expression of proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), in the ipsilateral TBI mouse cortex and hippocampus were significantly reduced by drug treatment. The increased expression of amyloid precursor protein (APP), phosphorylated Tau (p-Tau), phosphorylated glycogen synthase kinase 3 beta (pGSK3β) and p35/p25 subunits and the decreased expression of the pre-synaptic proteins, synaptophysin, synaptosome-associated protein of 25 kDa (SNAP25), and cysteine string protein alpha (α-CSP), in TBI mouse brain were also normalized by PF04458745 treatment. The improved locomotor function and working memory were partially mediated by activation of both cannabinoid (CB)1 and CB2 receptors, whereas the improvement on spatial learning and memory seemed to be CB1 receptor dependent. Interestingly, the blockage of PF04457845 on the reduced expression of synaptophysin, but not SNAP25 and α-CSP, was reversed by coadministration of the CB1 receptor antagonist. These results suggest that the therapeutic effect of PF04457845 is mediated by both cannabinoid receptor dependent and independent mechanisms, and selective inhibition of FAAH possesses a great potential for the treatment of TBI.

A Focus on the Beneficial Effects of Alpha Synuclein and a Re-Appraisal of Synucleinopathies.

Alpha synuclein (α-syn) belongs to a class of proteins which are commonly considered to play a detrimental role in neuronal survival. This assumption is based on the occurrence of a severe neuronal degeneration in patients carrying a multiplication of the α-syn gene (SNCA) and in a variety of experi-mental models, where overexpression of α-syn leads to cell death and neurological impairment. In these conditions, a higher amount of normally structured α-syn produces a damage, which is even worse com-pared with that produced by α-syn owning an abnormal structure (as occurring following point gene muta-tions). In line with this, knocking out the expression of α-syn is reported to protect from specific neurotox-ins such as 1-methyl, 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the present review we briefly dis-cuss these well-known detrimental effects but we focus on findings showing that, in specific conditions α-syn is beneficial for cell survival. This occurs during methamphetamine intoxication which is counteracted by endogenous α-syn. Similarly, the dysfunction of the chaperone cysteine-string protein-alpha leads to cell pathology which is counteracted by over-expressing α-syn. In line with this, an increased expression of α-syn protects against oxidative damage produced by dopamine. Remarkably, when the lack of α-syn is combined with a depletion of β- and γ- synucleins, alterations in brain structure and function occur. This review tries to balance the evidence showing a beneficial effect with the bulk of data reporting a detri-mental effect of endogenous α-syn. The specific role of α-syn as a chaperone protein is discussed to ex-plain such a dual effect.

The role of cysteine string protein alpha phosphorylation at SER10 and SER34 by PKCGAMMA for the presynaptic maintenance

Background: Protein kinase C (PKC) has been implicated in the control of neuronal cell survival. PKCγ-KO animal models showed symptoms of parkinsonian syndrome, including the dopaminergic neuronal loss in the Substantia Nigra. However, the PKCγ substrates responsible for the dopaminergic neuronal cell survival in vivo have not yet been elucidated. We previously found 10 phosphoproteins including Cysteine string protein alpha (CSPα) that had decreased phosphorylation levels in the striatum of PKCγ-KO mice.

The Role of Co-chaperones in Synaptic Proteostasis and Neurodegenerative Disease.

Synapses must be preserved throughout an organism's lifespan to allow for normal brain function and behavior. Synapse maintenance is challenging given the long distances between the termini and the cell body, reliance on axonal transport for delivery of newly synthesized presynaptic proteins, and high rates of synaptic vesicle exo- and endocytosis. Hence, synapses rely on efficient proteostasis mechanisms to preserve their structure and function. To this end, the synaptic compartment has specific chaperones to support its functions. Without proper synaptic chaperone activity, local proteostasis imbalances lead to neurotransmission deficits, dismantling of synapses, and neurodegeneration. In this review, we address the roles of four synaptic chaperones in the maintenance of the nerve terminal, as well as their genetic links to neurodegenerative disease. Three of these are Hsp40 co-chaperones (DNAJs): Cysteine String Protein alpha (CSPα; DNAJC5), auxilin (DNAJC6), and Receptor-Mediated Endocytosis 8 (RME-8; DNAJC13). These co-chaperones contain a conserved J domain through which they form a complex with heat shock cognate 70 (Hsc70), enhancing the chaperone's ATPase activity. CSPα is a synaptic vesicle protein known to chaperone the t-SNARE SNAP-25 and the endocytic GTPase dynamin-1, thereby regulating synaptic vesicle exocytosis and endocytosis. Auxilin binds assembled clathrin cages, and through its interactions with Hsc70 leads to the uncoating of clathrin-coated vesicles, a process necessary for the regeneration of synaptic vesicles. RME-8 is a co-chaperone on endosomes and may have a role in clathrin-coated vesicle endocytosis on this organelle. These three co-chaperones maintain client function by preserving folding and assembly to prevent client aggregation, but they do not break down aggregates that have already formed. The fourth synaptic chaperone we will discuss is Heat shock protein 110 (Hsp110), which interacts with Hsc70, DNAJAs, and DNAJBs to constitute a disaggregase. Hsp110-related disaggregase activity is present at the synapse and is known to protect against aggregation of proteins such as α-synuclein. Congruent with their importance in the nervous system, mutations of these co-chaperones lead to familial neurodegenerative disease. CSPα mutations cause adult neuronal ceroid lipofuscinosis, while auxilin mutations result in early-onset Parkinson's disease, demonstrating their significance in preservation of the nervous system.

Gene Therapy of Adult Neuronal Ceroid Lipofuscinoses with CRISPR/Cas9 in Zebrafish.

Adult-onset neuronal ceroid lipofuscinosis (ANCL), one of the neuronal ceroid lipofuscinosis (NCLs), is an inherited neurodegenerative disorder with progressive neuronal dysfunction. Recently, mutations in the DNAJC5 gene that encodes cysteine-string protein alpha (CSPα) have been reported to be associated with familial autosomal-dominant ANCL (AD-ANCL). This study constructed an ANCL transgenic zebrafish model expressing the human mutant DNAJC5 (mDNAJC5) gene under the control of a zebrafish neuron-specific promoter. To investigate whether gene therapy based on genome-editing technology could treat ANCL, a panel of TALEN and Cas9 nucleases was designed to disrupt the mDNAJC5 gene in this transgenic animal model. By screening these nucleases, it was found that one nuclease that targeted the 5' coding region efficiently alleviated mDNAJC5 protein aggregates in the affected neurons. Therefore, this study provides a gene therapy strategy via the use of the CRISPR/Cas9 system to treat neural genetic diseases.

A cluster of palmitoylated cysteines are essential for aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding cysteine string protein alpha (CSPα). The disease-causing mutations, which result in substitution of leucine-115 with an arginine (L115R) or deletion of the neighbouring leucine-116 (∆L116) in the cysteine-string domain cause CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the cysteine-string domain, however the regions of the mutant proteins that drive aggregation have not been determined. The importance of specific residues in the cysteine-string domain was investigated, revealing that a central core of palmitoylated cysteines is essential for aggregation of ANCL CSPα mutants. Interestingly, palmitoylated monomers of ANCL CSPα mutants were shown to be short-lived compared with wild-type CSPα, suggesting that the mutants either have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates. These findings provide new insight into the features of CSPα that promote aggregation in the presence of L115R/∆L116 mutations and reveal a change in the lifetime of palmitoylated monomers of the mutant proteins.

Clinically early-stage CSPα mutation carrier exhibits remarkable terminal stage neuronal pathology with minimal evidence of synaptic loss.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (AD-ANCL) is a multisystem disease caused by mutations in the DNAJC5 gene. DNAJC5 encodes Cysteine String Protein-alpha (CSPα), a putative synaptic protein. AD-ANCL has been traditionally considered a lysosomal storage disease based on the intracellular accumulation of ceroid material. Here, we report for the first time the pathological findings of a patient in a clinically early stage of disease, which exhibits the typical neuronal intracellular ceroid accumulation and incipient neuroinflammation but no signs of brain atrophy, neurodegeneration or massive synaptic loss. Interestingly, we found minimal or no apparent reductions in CSPα or synaptophysin in the neuropil. In contrast, brain homogenates from terminal AD-ANCL patients exhibit significant reductions in SNARE-complex forming presynaptic protein levels, including a significant reduction in CSPα and SNAP-25. Frozen samples for the biochemical analyses of synaptic proteins were not available for the early stage AD-ANLC patient. These results suggest that the degeneration seen in the patients with AD-ANCL reported here might be a consequence of both the early effects of CSPα mutations at the cellular soma, most likely lysosome function, and subsequent neuronal loss and synaptic dysfunction.

Evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer's disease.

BackgroundIn Alzheimer’s disease synapse loss precedes neuronal loss and correlates best with impaired memory formation. However, the mechanisms underlying synaptic degeneration in Alzheimer’s disease are not well known. Further, it is unclear why synapses in AD cerebellum are protected from degeneration. Our recent work on the cyclin-dependent kinase 5 activator p25 suggested that expression of the multifunctional presynaptic molecule cysteine string protein alpha (CSPalpha) may be affected in Alzheimer’s disease.ResultsUsing western blots and immunohistochemistry, we found that CSPalpha expression is reduced in hippocampus and superior temporal gyrus in Alzheimer’s disease. Reduced CSPalpha expression occurred before synaptophysin levels drop, suggesting that it contributes to the initial stages of synaptic degeneration. Surprisingly, we also found that CSPalpha expression is upregulated in cerebellum in Alzheimer’s disease. This CSPalpha upregulation reached the same level as in young, healthy cerebellum. We tested the idea whether CSPalpha upregulation might be neuroprotective, using htau mice, a model of tauopathy that expresses the entire wild-type human tau gene in the absence of mouse tau. In htau mice CSPalpha expression was found to be elevated at times when neuronal loss did not occur.ConclusionOur findings provide evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer’s disease. In the forebrain CSPalpha expression is reduced early in the disease and this may contribute to the initial stages of synaptic degeneration. In the cerebellum CSPalpha expression is upregulated to young, healthy levels and this may protect cerebellar synapses and neurons to survive. Accordingly, CSPalpha upregulation also occurs in a mouse model of tauopathy only at time when neuronal loss does not take place.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0096-z) contains supplementary material, which is available to authorized users.

Reduced expression of the presynaptic co-chaperone cysteine string protein alpha (CSPα) does not exacerbate experimentally-induced ME7 prion disease

Infection of mice with the ME7 prion agent results in well-characterised neuropathological changes, which includes vacuolation, neurodegeneration and synaptic degeneration. Presynaptic dysfunction and degeneration is apparent through the progressive reduction in synaptic vesicle proteins and eventual loss of synapses. Cysteine string protein alpha (CSPα), which regulates refolding pathways at the synapse, exhibits an early decline during chronic neurodegeneration implicating it as a mediator of disease mechanisms. CSPα null mice develop a progressive neuronal dysfunction through disruption of the integrity of presynaptic function. In this study, we investigated whether reduced expression of CSPα would exacerbate ME7 prion disease. Wild type (+/+) and heterozygous (+/−) mice, which express about a ∼50% reduction in CSPα, were used as a distinct genetic background on which to impose prion disease. +/+ and +/ − mice were inoculated with brain homogenate from either a normal mouse brain (NBH) or from the brain of a mouse which displayed clinical signs of prion disease (ME7). Behavioural tests, western blotting and immunohistochemistry, which resolve key elements of synaptic dysfunction, were used to assess the effect of reduced CSPα on disease. Behavioural tests revealed no change in the progression of disease in ME7–CSPα +/− animals compared to ME7–CSPα +/+ animals. In addition, the accumulation of misfolded PrPSc, the diseased associated gliosis or synaptic loss were not different. Thus, the misfolding events that generate synaptic dysfunction and lead to synaptic loss are unlikely to be mediated by a disease associated decrease in the refolding pathways associated with CSPα.

Oligomerization of Cysteine String Protein alpha mutants causing adult neuronal ceroid lipofuscinosis

Cysteine String Protein alpha (CSPα) is a palmitoylated, synaptic vesicle co-chaperone that is essential for neuroprotection. Two mutations in CSPα — L115R and L116Δ — cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly-inherited neurodegenerative disease. To elucidate the pathogenesis of ANCL, the intrinsic biochemical properties of human wildtype (WT) and disease mutant CSPα were examined. Mutant proteins purified from Escherichia coli exhibited high potency to oligomerize in a concentration, temperature, and time dependent manner, with L115R possessing the greatest potency. When freshly purified, ANCL mutant proteins displayed normal co-chaperone activity and substrate recognition similar to WT. However, co-chaperone activity was impaired for both CSPα mutants upon oligomerization. When WT and mutant CSPα were mixed together they co-oligomerized leading to an overall decrease of co-chaperone activity. The oligomerization properties of ANCL mutants were faithfully replicated in HEK 293T cells. Interestingly, the oligomers were covalently tagged by ubiquitination instead of palmitoylation. Taken together, ANCL mutations result in both a gain and partial loss-of-function.

Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol.

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

Mutations in DNAJC5, Encoding Cysteine-String Protein Alpha, Cause Autosomal-Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSPα). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

Palmitoylation and Testis-Enriched Expression of the Cysteine-String Protein β Isoform

Cysteine-string protein alpha (CSPalpha) is a DnaJ chaperone that is associated with secretory vesicles in diverse cell types. The cysteine-string region is the signature domain of CSPalpha and contains 14 closely spaced cysteines, the majority of which are palmitoylated; this post-translational modification mediates stable membrane attachment. CSPalpha has been proposed to function in regulated exocytosis pathways throughout the body and has an additional neuroprotective function. Two novel CSP isoforms, beta and gamma, were identified recently, although the expression profile, properties, and functions of these proteins are not clear and in some cases are subject to debate. Here, we report that CSPbeta is enriched in rat testis and was not detected in any other tissue that was examined, including brain. Although the cysteine-string domain of CSPbeta is distinct from that found in CSPalpha, the endogenous beta isoform expressed in testis is membrane-associated and palmitoylated. However, in agreement with earlier work, we find that the palmitoylation efficiency of CSPbeta is reduced compared with that of CSPalpha. Subsequent analysis of chimeric proteins reveals that regions upstream of the cysteine-string domains of CSPalpha and CSPbeta underlie this difference in palmitoylation efficiency between the two isoforms.

α-Synuclein Cooperates with CSPα in Preventing Neurodegeneration

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.