Diffusely abnormal white matter in clinically isolated syndrome is associated with parenchymal loss and elevated neurofilament levels.

We characterized the frequency of diffusely abnormal white matter (DAWM) across a broad spectrum of multiple sclerosis (MS) participants. 35% of clinically isolated syndrome (CIS), 57% of relapsing remitting and 64% of secondary progressive MS participants demonstrated DAWM. CIS with DAWM had decreased cortical thickness, higher lesion load and a higher concentration of serum neurofilament light chain compared to CIS without DAWM. DAWM may be useful in identifying CIS patients with greater injury to their brains. Larger and longitudinal studies are warranted.

Technique and preliminary findings for in vivo quantification of brain motion during injurious head impacts.

Computational models of the human brain are widely used in the evaluation and development of helmets and other protective equipment. These models are often attempted to be validated using cadaver tissue displacements despite studies showing neural tissue degrades quickly after death. Addressing this limitation, this study aimed to develop a technique for quantifying living brain motion in vivo using a closed head impact animal model of traumatic brain injury (TBI) called CHIMERA. We implanted radiopaque markers within the brain of three adult ferrets and resealed the skull while the animals were anesthetized. We affixed additional markers to the skull to track skull kinematics. The CHIMERA device delivered controlled, repeatable head impacts to the head of the animals while the impacts were fluoroscopically stereo-visualized. We observed that 1.5 mm stainless steel fiducials (∼8 times the density of the brain) migrated from their implanted positions while neutral density targets remained in their implanted position post-impact. Brain motion relative to the skull was quantified in neutral density target tests and showed increasing relative motion at higher head impact severities. We observed the motion of the brain lagged behind that of the skull, similar to previous studies. This technique can be used to obtain a comprehensive dataset of in vivo brain motion to validate computational models reflecting the mechanical properties of the living brain. The technique would also allow the mechanical response of in vivo brain tissue to be compared to cadaveric preparations for investigating the fidelity of current human computational brain models.

modCHIMERA: a novel murine closed-head model of moderate traumatic brain injury.

Traumatic brain injury is a major source of global disability and mortality. Preclinical TBI models are a crucial component of therapeutic investigation. We report a tunable, monitored model of murine non-surgical, diffuse closed-head injury-modCHIMERA-characterized by impact as well as linear and rotational acceleration. modCHIMERA is based on the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA) platform. We tested this model at 2 energy levels: 1.7 and 2.1 Joules-substantially higher than previously reported for this system. Kinematic analysis demonstrated linear acceleration exceeding injury thresholds in humans, although outcome metrics tracked impact energy more closely than kinematic parameters. Acute severity metrics were consistent with a complicated-mild or moderate TBI, a clinical population characterized by high morbidity but potentially reversible pathology. Axonal injury was multifocal and bilateral, neuronal death was detected in the hippocampus, and microglial neuroinflammation was prominent. Acute functional analysis revealed prolonged post-injury unconsciousness, and decreased spontaneous behavior and stimulated neurological scores. Neurobehavioral deficits were demonstrated in spatial learning/memory and socialization at 1-month. The overall injury profile of modCHIMERA corresponds with the range responsible for a substantial portion of TBI-related disability in humans. modCHIMERA should provide a reliable platform for efficient analysis of TBI pathophysiology and testing of treatment modalities.

Cholesterol at the crossroads: Alzheimer's disease and lipid metabolism.

Alzheimer's Disease (AD) is a devastating disease that affects millions of elderly persons. Despite years of intense investigations, genetic risk factors that affect the majority of AD cases have yet to be determined. Recent studies suggest that cholesterol metabolism has integral part in AD pathogenesis, suggesting that genes that regulate lipid metabolism may also play roles in AD. This review will first describe emerging evidence that links cholesterol to the mechanisms thought to underlie AD. Based on this rationale, candidate genes located in regions implicated in AD that have roles in lipid metabolism will then be discussed.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Human ABCA1 BAC transgenic mice show increased high density lipoprotein cholesterol and ApoAI-dependent efflux stimulated by an internal promoter containing liver X receptor response elements in intron 1.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

Mutant huntingtin enhances excitotoxic cell death.

Evidence suggests overactivation of NMDA-type glutamate receptors (NMDARs) contributes to selective degeneration of medium-sized spiny striatal neurons in Huntington's disease (HD). Here we determined whether expression of huntingtin containing the polyglutamine expansion augments NMDAR-mediated excitotoxicity. HEK293 cells coexpressing mutant huntingtin (htt-138Q) and either NR1A/NR2A- or NR1A/NR2B-type NMDARs exposed to 1 mM NMDA showed a significant increase in excitotoxic cell death compared to controls (cells coexpressing htt-15Q or GFP), but the difference was larger for NR1A/NR2B. Moreover, agonist-dependent cell death showed apoptotic features for cells coexpressing htt-138Q and NR1A/NR2B, but not for cells expressing htt-138Q and NR1A/NR2A. Further, NR1A/NR2B-mediated apoptosis was not seen with coexpression of an N-terminal fragment of mutant htt. Since NR1A/NR2B is the predominant NMDAR subtype in neostriatal medium-sized spiny neurons, enhancement of NMDA-induced apoptotic death in NR1A/NR2B-expressing cells by full-length mutant htt may contribute to selective neurodegeneration in HD.

Huntington disease: new insights on the role of huntingtin cleavage.

Huntington Disease (HD) results from polyglutamine expansion within the N-terminus of huntingtin. We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) human huntingtin in a developmentally appropriate and tissue-specific manner identical to the pattern of expression of endogenous huntingtin. YAC46 and YAC72 mice show early electrophysiological abnormalities indicating neuronal cytoplasmic dysfunction prior to developing nuclear inclusions or neurodegeneration. YAC72 mice display a hyperkinetic movement disorder by 7 months of age, and have evidence for selective and specific degeneration of medium spiny neurons in the lateral striatum by 12 months of age. A key molecular feature of pathology of these YAC72 mice is cleavage of huntingtin in the cytoplasm following by translocation of the resulting huntingtin N-terminal fragments into the nucleus of striatal neurons. Increasing nuclear localization of huntingtin N-terminal fragments within medium spiny neurons of the striatum occurs concomitantly with the onset of selective neurodegeneration. Because huntingtin is a caspase substrate and truncated huntingtin fragments are toxic in vitro, inhibiting caspase cleavage of huntingtin may be of potential therapeutic benefit in HD. We show that caspase inhibitors eliminate huntingtin cleavage in cells and protects them from an apoptotic stress. We also identify caspase-6 and caspase-3 cleavage sites in huntingtin and demonstrate that neuronal and non-neuronal cells expressing a caspase-resistant huntingtin with an expanded polyglutamine tract are less susceptible to apoptosis and aggregate formation. These results suggest that caspase cleavage of huntingtin may be a crucial step in aggregate formation and neurotoxicity in HD.

Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain.

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.

Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells.

Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a site-directed htt mutant that is resistant to caspase-3 cleavage at positions 513 and 530 and to caspase-6 cleavage at position 586. In contrast to cleavable htt, caspase-resistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease.

Caspases and neurodegeneration: on the cutting edge of new therapeutic approaches.

Unregulated apoptosis underlies many pathological conditions, including neurodegenerative diseases. In this review, we focus on the role of cysteine aspartate-specific proteases (caspase) activity in Huntington disease (HD) and Alzheimer disease (AD) as two representative neurodegenerative disorders that normally manifest in mid- to late-life. Caspases appear to be involved in the molecular pathology of HD by directly cleaving huntingtin and generating toxic protein fragments containing the polyglutamine tract, and by being recruited and activated by polyglutamine-containing aggregates composed mainly of truncated huntingtin fragments. Several proteins involved in AD, including beta-amyloid precursor protein (APP) and presenilins (PSs), are also cleaved by caspases. For APP, caspase cleavage may contribute to toxicity by generating toxic fragments or by shifting APP processing toward an amyloidogenic pathway. For PSs, caspase cleavage disables antiapoptotic functions attributed to PS C-terminal fragments. These observations suggest that caspases actively contribute to the molecular pathogenesis of these diseases and support the development of caspase inhibitors as potential therapeutic approaches for chronic neurodegenerative disorders.

Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity.

Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin-1 (DRPLA), ataxin-3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin-1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin-1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin-1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin-1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin-1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutamine-containing proteins may be a feasible therapeutic strategy to prevent cell death.

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity.

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.

Recent insights into the molecular pathogenesis of Huntington disease.

Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the HD gene resulting in expression of an uninterrupted polyglutamine stretch within the N-terminus of its protein product huntingtin (htt). In this article we review the clinical, genetic, and neuropathological features of HD and discuss recent insights into the pathogenesis of HD. Examining the role of CAG repeat size on age of onset and penetrance in HD using a refined database of human HD patients has provided further support for the importance of the CAG repeat in the pathogenesis of HD and information leading to a predictive model for the likelihood of being affected by a specific age for a particular CAG expansion. In a YAC transgenic mouse model that replicates key elements of the HD phenotype, the development of selective striatal neurodegeneration is coincident with cleavage of htt and translocation of the N-terminal htt fragment into the nucleus. We also review in vitro evidence that htt is a substrate for cleavage by a group of cysteine proteases involved in apoptotic death-the caspases, and that caspase cleavage of htt results in the generation of a toxic N-terminal fragment. Inhibiting caspase cleavage of huntingtin eliminates the toxicity of the mutant htt protein. These results suggest that cleavage of huntingtin resulting in production of a truncated N-terminal fragment may be a crucial step in the pathogenesis of Huntington disease and that inhibition of this process may be a potential therapeutic strategy for this currently untreatable disorder.

Quiescence versus apoptosis: Myc abundance determines pathway of exit from the cell cycle.

When exposed to diverse growth conditions in vitro, cells can respond by entering states of proliferation, quiescence, differentiation or apoptosis. While the choices among these states can be influenced by proto-oncogene expression, how these disparate outcomes are achieved remains poorly understood. To address these issues, we have generated rodent fibroblast cell lines that harbor a human c-myc gene under the control of a tetracycline-regulated promoter. When Myc-induced cells are deprived of serum growth factors, they rapidly become apoptotic with the onset of apoptosis preceded by a large, transient increase in cdk2 kinase activity that is associated with the induction of cdc25A phosphatase and the later accumulation of p27Kip1 kinase inhibitor. Surprisingly, serum starvation in the absence of myc overexpression, (which leads to quiescence instead of apoptosis) also causes a marked transient elevation in cdk2 kinase activity, an induction of cdc25A and a delayed increase in p27Kip1. Transient elevations in cdk2 kinase activity and cdc25A abundance are required for cell cycle progression, but it is evident that these changes also precede entry to either apoptosis or quiescence in serum-starved cells. These findings suggest that the pathways to both quiescence and apoptosis share regulatory machinery with cell cycle control mechanisms. In addition, the abundance of Myc protein can be critical in the choices among these cellular states.

The influence of huntingtin protein size on nuclear localization and cellular toxicity.

Huntington disease is an autosomal dominant neurodegenerative disorder caused by the pathological expansion of a polyglutamine tract. In this study we directly assess the influence of protein size on the formation and subcellular localization of huntingtin aggregates. We have created numerous deletion constructs expressing successively smaller fragments of huntingtin and show that these smaller proteins containing 128 glutamines form both intranuclear and perinuclear aggregates. In contrast, larger NH2-terminal fragments of huntingtin proteins with 128 glutamines form exclusively perinuclear aggregates. These aggregates can form in the absence of endogenous huntingtin. Furthermore, expression of mutant huntingtin results in increased susceptibility to apoptotic stress that is greater with decreasing protein length and increasing polyglutamine size. As both intranuclear and perinuclear aggregates are clearly associated with increased cellular toxicity, this supports an important role for toxic polyglutamine-containing fragments forming aggregates and playing a key role in the pathogenesis of Huntington disease.

Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract.

The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin-1, ataxin-3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the full-length proteins. Our results suggest that by generation of truncated polyglutamine-containing proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases.

Of molecular interactions, mice and mechanisms: new insights into Huntington's disease.

Huntington's disease is caused by expansion of a CAG trinucleotide beyond 35 repeats within the coding region of a novel gene. Recently, new insights into the relationship between CAG expansion in the HD gene and pathological mechanisms have emerged. These include a more precise understanding of the relationship between CAG repeat length and age of onset, progress in transgenic and excitotoxic animal models, identification of a novel huntington-interacting protein, and intriguing connections between huntington and the apoptotic machinery. We have combined many of these new findings into a model that suggests mechanisms and predicts outcomes by which the pathogenesis of Huntington's disease may be initiated. The development of appropriate in-vitro and animal models for Huntington's disease will allow the validity of this model to be tested.

Toward understanding the molecular pathology of Huntington's disease.

Huntington's Disease (HD) is caused by expansion of a CAG trinucleotide beyond 35 repeats within the coding region of a novel gene. Recently, new insights into the relationship between CAG expansion in the HD gene and pathological mechanisms have emerged. Survival analysis of a large cohort of affected and at-risk individuals with CAG sizes between 39 and 50 repeats have yielded probability curves of developing HD symptoms and dying of HD by a certain age. Animals transgenic for the first exon of huntingtin with large CAG repeats lengths have been reported to have a complex neurological phenotype that bears interesting similarities and differences to HD. The repertoire of huntingtin-interacting proteins continues to expand with the identification of HIP1, a protein whose yeast homologues have known functions in regulating events associated with the cytoskeleton. The ability of huntingtin to interact with two of its four known protein partners appears to be influenced by CAG length. Caspase 3 (apopain), a key cysteine protease known to play a seminal role in neural apoptosis, has also been demonstrated to specifically cleave huntingtin in a CAG length-dependent manner. Many of these features are combined in a model suggesting mechanisms by which the pathogenesis of HD may be initiated. The development of appropriate in vitro and animal models for HD will allow the validity of these models to be tested.