Individuals with Parkinson's disease (PD) typically receive a diagnosis once they have developed motor symptoms, at which point there is already significant loss of substantia nigra dopamine neurons, ...α-synuclein accumulation in surviving neurons, and neuroinflammation. Consequently, the point of clinical presentation may be too late to initiate disease-modifying therapy. In contrast to this clinical reality, animal models often involve acute neurodegeneration and potential therapies are tested concurrently or shortly after the pathogenic insult has begun rather than later when diagnostic clinical symptoms emerge. Therefore, we sought to develop a model that reflects the clinical situation more accurately. Middle-aged rats (7-9 months-old) received a single daily intraperitoneal injection of rotenone for 5 consecutive days and were observed over the next 8-9 months. Rotenone-treated rats showed transient motor slowing and postural instability during exposure but recovered within 9 days of rotenone cessation. Rats remained without behavioral deficits for 3-4 months, then developed progressive motor abnormalities over the ensuing months. As motor abnormalities began to emerge 3 months after rotenone exposure, there was significant loss of nigral dopaminergic neurons and significant microglial activation. There was delayed accumulation of α-synuclein in neurons of the substantia nigra and frontal cortex, which was maximal at 9 months post-rotenone. In summary, a brief temporally-remote exposure to rotenone causes delayed and progressive behavioral and neuropathological changes similar to Parkinson's disease. This model mimics the human clinical situation, in which pathogenesis is well-established by the time diagnostic motor deficits appear. As such, this model may provide a more relevant experimental system in which to test disease-modifying therapeutics.
DYT1 dystonia is caused by mutation of the TOR1A gene, resulting in the loss of a single glutamic acid residue near the carboxyl terminal of TorsinA. The neuronal functions perturbed by TorsinAΔE are ...a major unresolved issue in understanding the pathophysiology of dystonia, presenting a critical roadblock to developing effective treatments. We identified and characterized the zebrafish homologue of TOR1A, as a first step towards elucidating the functions of TorsinA in neurons, in vivo, using the genetically-manipulable zebrafish model. The zebrafish genome was found to contain a single alternatively-spliced tor1 gene, derived from a common ancestral locus shared with the dual TOR1A and TOR1B paralogues found in tertrapods. tor1 was expressed ubiquitously during early embryonic development and in multiple adult tissues, including the CNS. The 2.1 kb tor1 mRNA encodes Torsin1, which is 59% identical and 78% homologous to human TorsinA. Torsin1 was expressed as major 45 kDa and minor 47 kDa glycoproteins, within the cytoplasm of neurons and neuropil throughout the CNS. Similar to previous findings relating to human TorsinA, mutations of the ATP hydrolysis domain of Torsin1 resulted in relocalization of the protein in cultured cells from the endoplasmic reticulum to the nuclear envelope. Zebrafish embryos lacking tor1 during early development did not show impaired viability, overt morphological abnormalities, alterations in motor behavior, or developmental defects in the dopaminergic system. Torsin1 is thus non-essential for early development of the motor system, suggesting that important CNS functions may occur later in development, consistent with the critical time window in late childhood when dystonia symptoms usually emerge in DYT1 patients. The similarities between Torsin1 and human TorsinA in domain organization, expression pattern, and cellular localization suggest that the zebrafish will provide a useful model to understand the neuronal functions of Torsins in vivo.
The catecholamine neurotransmitter dopamine has the potential to act as an endogenous neurotoxin when its vesicular sequestration is dysregulated. Despite postmortem analyses from patients with ...Parkinson's disease that demonstrate decreased vesicular sequestration of dopamine with a corresponding increase in dopamine metabolism, dopamine's contribution to nigrostriatal dopaminergic degeneration in Parkinson's disease has been debated. Here, we present a new in vivo model demonstrating the induction of Parkinson's disease-associated pathogenic mechanisms of degeneration resulting from acquired dysregulation of dopamine sequestration in nigrostriatal dopaminergic neurons in adult rats. Utilizing adeno-associated virus (serotype 2), viral-mediated small-hairpin RNA interference of endogenous vesicular monoamine transporter 2 (VMAT2) expression resulted in a loss of VMAT2 protein expression in transduced dopaminergic cell bodies in the substantia nigra with a corresponding loss of VMAT2 protein within the striatal terminals. The loss of VMAT2 resulted in an accumulation of cytosolic dopamine and subsequent increased dopamine metabolism, deficits in dopamine-mediated behaviors, and degeneration of nigrostriatal dopaminergic neurons that was rescued through reintroduction of exogenous VMAT2, demonstrating that the toxicity was specific to the loss of VMAT2. Analysis of parkinsonian pathogenic mechanisms of degeneration identified oxidative damage, activation of Parkinson's disease-associated kinase LRRK2, and the formation of aberrant α-synuclein. This model demonstrates that a progressive acquired loss of VMAT2 expression in adulthood is sufficient to induce Parkinson's disease-associated pathogenic mechanisms of degeneration and provides a new model to further investigate the consequences of cytosolic dopamine.
Frontotemporal degeneration (FTD) is a common cause of dementia for which there are currently no approved therapies. Over the past decade, there has been an explosion of knowledge about the biology ...and clinical features of FTD that has identified a number of promising therapeutic targets as well as animal models in which to develop drugs. The close association of some forms of FTD with neuropathological accumulation of tau protein or increased neuroinflammation due to progranulin protein deficiency suggests that a drug's success in treating FTD may predict efficacy in more common diseases such as Alzheimer's disease. A variety of regulatory incentives, clinical features of FTD such as rapid disease progression, and relatively pure molecular pathology suggest that there are advantages to developing drugs for FTD as compared with other more common neurodegenerative diseases such as Alzheimer's disease. In March 2011, the Frontotemporal Degeneration Treatment Study Group sponsored a conference entitled “FTD, the Next Therapeutic Frontier,” which focused on preclinical aspects of FTD drug development. The goal of the meeting was to promote collaborations between academic researchers and biotechnology and pharmaceutical researchers to accelerate the development of new treatments for FTD. Here we report the key findings from the conference, including the rationale for FTD drug development; epidemiological, genetic, and neuropathological features of FTD; FTD animal models and how best to use them; and examples of successful drug development collaborations in other neurodegenerative diseases.
Loss-of-function mutations in the human
PARK7 gene, encoding DJ-1, are a rare cause of autosomal recessive Parkinson's disease (ARPD). To facilitate generation of a novel vertebrate model, in which ...to examine the biochemical functions of DJ-1
in vivo, we cloned and characterized the zebrafish orthologue of DJ-1 (zDJ-1). The 0.95 kb zDJ-1 mRNA is expressed in adult zebrafish brain, muscle and gut, and in the embryo from 24 h post-fertilization. The zDJ-1 transcript encodes a 19.8 kDa, 189 amino acid protein, which is 83% identical to human DJ-1. Residues thought to be functionally important sites of post-translational modification in human DJ-1, and critical positions affected by pathogenic missense mutations in ARPD patients, are conserved in zDJ-1. The 14 kb zDJ-1 gene contains six exons and is located on zebrafish chromosome 8; the structure of the gene is highly homologous to human DJ-1, except that there are no alternatively spliced non-coding 5′ exons. The single zDJ-1 first exon shows 5′ end heterogeneity, reflecting multiple transcription start sites. In the adult zebrafish brain, zDJ-1 immunoreactivity was prominent in the cytoplasm of most neurons, and in the neuropil, but was less evident within white matter tracts, consistent with neuronal somatic and dendritic localization. Dopaminergic neurons in each of the major forebrain and diencephalic TH-positive cell groups expressed zDJ-1. These studies show that zDJ-1 is very similar to human DJ-1 and delineate essential resources, allowing further examination of the function and regulation of DJ-1, using the zebrafish as a model.
Duchenne muscular dystrophy (DMD) is a fatal childhood disease caused by mutations that abolish the expression of dystrophin
in muscle. Utrophin is a paralogue of dystrophin and can functionally ...replace it in skeletal muscle. A method to induce utrophin
up-regulation in muscle should therefore be therapeutically useful in DMD. We have previously shown that there are two full-length
utrophin mRNA species: A and B. Here we describe the generation and characterization of antibodies specific to A- and B-utrophin.
We show that both mRNA isoforms are translated into full-length proteins, which have very different expression patterns. B-utrophin
is expressed in vascular endothelial cells; A-utrophin is expressed at the neuromuscular junction, choroid plexus, pia mater,
and renal glomerulus. We have analyzed the expression of A- and B-utrophin protein and RNA in dystrophin-deficient tissues.
We conclude that (i) the previously described expression patterns of utrophin represent a composite of A- and B-utrophin,
(ii) A- but not B-utrophin is up-regulated in dystrophin-deficient striated muscle, and (iii) this up-regulation occurs post-transcriptionally
with an additional transcriptional component in skeletal muscle. These results have important implications for understanding
the biology of utrophin and are crucial for future studies aiming to effect its therapeutic up-regulation in DMD patients.
Frontotemporal degeneration (FTD) encompasses a spectrum of related neurodegenerative disorders with behavioral, language, and motor phenotypes for which there are currently no effective therapies. ...This is the second of two articles that summarize the presentations and discussions that occurred at two symposia in 2011 sponsored by the Frontotemporal Degeneration Treatment Study Group, a collaborative group of academic and industry researchers that is devoted to developing treatments for FTD. This article discusses the current status of FTD clinical research that is relevant to the conduct of clinical trials, and why FTD research may be an attractive pathway for developing therapies for neurodegenerative disorders. The clinical and molecular features of FTD, including rapid disease progression and relatively pure molecular pathology, suggest that there are advantages to developing drugs for FTD as compared with other dementias. FTD qualifies as orphan indication, providing additional advantages for drug development. Two recent sets of consensus diagnostic criteria will facilitate the identification of patients with FTD, and a variety of neuropsychological, functional, and behavioral scales have been shown to be sensitive to disease progression. Moreover, quantitative neuroimaging measurements demonstrate progressive brain atrophy in FTD at rates that may surpass Alzheimer's disease. Finally, the similarities between FTD and other neurodegenerative diseases with drug development efforts already underway suggest that FTD researchers will be able to draw on this experience to create a road map for FTD drug development. We conclude that FTD research has reached sufficient maturity to pursue clinical development of specific FTD therapies.
Abstract only
Progressive supranuclear palsy (PSP) is a common neurodegenerative disorder causing progressive motor, oculomotor, bulbar, and cognitive deficits. Current treatments do not arrest ...disease progression and the prognosis for functional independence and survival is correspondingly poor. Pathologically, PSP is characterized by accumulation of insoluble hyperphosphorylated forms of the 4‐repeat isoform of the microtubule associated protein tau (4R‐Tau) and subsequent loss of CNS neurons. Convergent lines of evidence suggest that 4R‐Tau is mechanistically involved in the neurodegeneration of PSP. Clinical and pathological PSP phenocopies can result from splice site mutations in the
MAPT
gene causing over‐expression of 4R‐Tau. Furthermore, PSP is strongly associated with the H1
MAPT
haplotype that gives rise to relative over‐production of 4R‐Tau. Consequently, compounds that prevent neurodegeneration caused by 4R‐Tau
in vivo
may be effective therapeutic agents in PSP.
The long‐term goal of our work is to develop effective treatments for progressive supranuclear palsy (PSP). Development of therapies for PSP and other tauopathies has been limited by lack of animal models that allow discovery‐based approaches, for example screening candidate chemical modifiers against disease‐relevant phenotypes
in vivo
. The zebrafish is a genetically and chemically tractable vertebrate model that is particularly suited to drug discovery and comparative efficacy testing and is being used increasingly for studying human neurological diseases. The objective of this study was to develop a zebrafish model of PSP and to carry out a phenotype based chemical modifier screen
in vivo
.
We generated transgenic zebrafish that express human 4R‐Tau in CNS neurons. Tau transgenic zebrafish replicated many clinic‐pathological features of PSP, including: hypokinesia; oculomotor deficits; somato‐dendritic accumulation of hyperphosphorylated tau; neuronal apoptosis; and impaired survival. Importantly, these phenotypes were quantitative, replicable and occured rapidly allowing their use as endpoints in a phenotype‐driven screen. We tested over 200 bioactive compounds to identify chemicals that rescued motor function in the 4R‐Tau zebrafish, using a custom automated video tracking and motion analysis method. We identified several ‘hits’ that rescued motor function and showed activity in subsequent verification assays.
Our completion of the first unbiased phenotype‐based chemical modifier screen in a vertebrate tauopathy model provides proof‐of‐concept that drug discovery can be carried out
in vivo
by assaying disease‐relevant neurological deficits. Importantly our screen has so far identified several candidates for further development as possible tau therapeutics.
Support or Funding Information
US Department of Veterans' Affairs (BX003168‐01)
NINDS (NS080881)
CurePSP (441‐05, 468‐08)
Pittsburgh Foundation (AD201577642)
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