Huntington's disease (HD) (Huntington's chorea, Huntington disease) . How is the cost effectiveness of alternative diagnostic methods to. Disease Primer Molecular Biology and Genetics of Huntington Disease (HD) Signs and Symptoms Biomarkers. Huntington's disease (HD) is a prototypical neurodegenerative disease in which there is selective neuronal degeneration, .. α-Tocopherol (Vitamin E).
disease 3.1.3. Huntington’s
Histopathologically, HD is characterised by premature death particularly of medium-sized mainly GABA-ergic striatal neurons but large interneurons are mostly spared e.
Regardless of the development of reactive gliosis within the striatum, loss of the grey matter is extensive and results in the compensatory enlargement of lateral brain ventricles. Severity of the degeneration is evaluated using grading system proposed by Vonsattel and coauthors [ 2 ], which classifies HD into five grades 0—4.
In grade 0, patients have already clinical symptoms; however, neuronal loss in the head of the caudate nucleus CN can be detected only on microscopic level.
In grade 1, neuronal loss is accompanied by reactive gliosis, which is evident primarily in the head and tail, less in the body of CN. Grade 2 is characterised by mild to moderated striatal atrophy. In grade 3, due to the progression of atrophy, the medial outline of the CN becomes straight.
Moreover, brains from patients with late onset of clinical symptoms might show changes occurring in usual aging in addition to those observed in HD e. Genetic mutation on short arm of chromosome 4, which causes HD, was discovered in [ 4 ]. Lately, in , gene IT15 interesting transcript 15 , which codes unstable protein huntingtin htt comprising variable number of CAG repeats, was identified [ 5 ].
Such molecular defect is based on the expansion of this triplet that codes amino acid glutamine. Therefore, HD can be included into the group of polyglutaminopathies. Mutant form of huntingtin mhtt comprises up to 40 repeats and individuals with 36—39 CAG repeats are in risk of developing adult late-onset form of HD.
Juvenile form of HD develops in patients with 55 and more repeats e. Although wild-type huntingtin is expressed in all cell types, with the highest concentration in the brain [ 7 ], its functions are not yet fully understood [ 8 ]. Expanded polyglutamine forms intracellular deposits, particularly in a form of intranuclear and neuropil aggregates e.
It is evident that mhtt displays specific toxicity to striatal mainly GABA-ergic , less cortical neurons. However, the role of mhtt in the pathogenesis of HD appears highly controversial, ranging from being essential in pathogenesis of the disease e. Moreover, mhtt also accumulates in nuclei of astrocytes causing their dysfunction, particularly in relation to glutamate uptake. This may further promote vulnerability, especially of striatal medium-sized spiny neurons, to the excitotoxic damage [ 12 ].
Therefore, turning our attention to glutamate uptake in astrocytes might be of particular importance for preventing glutamate excitotoxicity in HD [ 12 , 13 ]. Nevertheless, the presence of mhtt in neural cells is not the only mechanism, which is involved in HD pathogenesis. Metabolic and mitochondrial dysfunctions, oxidative and nitrative stress, and also apoptosis play an important role as well e.
Generation of transgenic mice as well as transgenic rats advanced significantly the understanding of HD pathology. Indeed, over 20 different rodent models of this disease have been generated to date for the review see [ 15 ]. These models of HD are characterized by early onset and fast progression of behavioural deficits later also by motor dysfunction and presence of intranuclear polyglutamine polyQ inclusions 4 weeks postnatally , but without the evident reduction of neurons even in aged mice [ 16 , 19 ].
Therefore, they simulate rather juvenile than adult form of HD. Due to relatively smaller number of CAG repeats, this model exhibits a high degree of similarity to the late-onset form of HD.
These rats survived up to 24 months and exhibited slow cognitive decline and not as much of motor deficit e. Neuropil aggregates of polyglutamine and typical intranuclear inclusions in neurons appeared in the brain approximately at 6—9 months of age [ 9 ].
Despite the fact that tgHD51 rats displayed enlarged lateral brain ventricles, stereological analysis revealed only a subtle decrease in the number of neurons in the striatum, while no changes were observed in number of neurons in the frontal cortical layer V of month-old tgHD51 rats when compared with age-matched wild-type controls [ 24 ].
Astrocytes are the most numerous type of glial cells in mammalian CNS. Currently, they are considered as highly active cellular component of the CNS parenchyma with functional pleiotropy essential for neuronal survival and function e. It has been suggested that enhanced release of glutamate and other substances may represent an early event in a number of, if not all, neurodegenerative diseases e.
The communication between neurons and local blood flow mediated by astrocytes is elementary for the maintenance of functional microenvironment in the grey matter of the entire CNS parenchyma; therefore the term neuronal-glial-vascular unit is used [ 30 ]. On the other hand, under pathological conditions, the perivascular end-feet can restrict transport or diffusion across the blood-brain interface [ 31 ]. Glial fibrillary acidic protein GFAP [ 32 ] is essential for immunohistochemical identification of astrocytes.
However, GFAP is densely expressed only within the cell body and larger processes of astrocytes, unlike the numerous fine processes representing the majority of the total volume of the astrocytes, which are GFAP-negative [ 30 ]. Any type of the CNS injury primarily the acute damage initiates morphological changes of some astrocytes, which become reactive e. They are hypertrophic with longer and thicker main processes and increased expression of GFAP due to the formation of bundles of gliofilaments e.
In the acute phase of reaction, astrocytes also reexpress intermediate filaments significant for glial precursors—nestin and vimentin e. NG2 glia polydendrocytes or synantocytes represent a fourth type of glia in the CNS e. They exist abundantly in both grey and white matter of the mature CNS in rodents as well as human. They constitute the major group of cells undergoing mitosis in the adult rodent brain and are almost as numerous as astrocytes [ 42 ].
NG2 cells are primarily described as the precursors of myelinating oligodendrocytes OLPs. However, many of the NG2 cells remain in the NG2-positive state for a significant time and have a unique capacity to communicate with nearby cells, forming multiple contacts with astrocytes, microglia, oligodendrocytes, and even neurons [ 43 ].
In human brain, significant morphological changes related to the progression of pathology were studied particularly in multiple sclerosis and gliomas [ 44 ]. Microglia, the immunocompetent highly motile cells of the CNS, are extremely plastic and undergo a variety of structural changes based on their location and current role [ 45 ].
In the grey matter, the most frequent is ramified form, which express protein Iba1 ionized calcium-binding adapter molecule 1 also known as AIF-1 allograft inflammatory factor 1. The density of this marker significantly increases with activation of cells. It is obvious that the activation of microglia is a basic mechanism in the defence of the CNS, also in relation to neurodegenerative processes e.
Although the role of microglia in neurodegeneration is still controversial, it is evident that in human brain they are activated in early stages of NDP of different phenotype, primarily in HD e. It is possible that microglia transform to phagocytes and target neurons as the disease progresses but appear to be dysfunctional with increasing amounts of ingested debris [ 48 ].
It is commonly known that the neurodegenerative process of HD phenotype is a chronic process, morphologically characterized by the progressive degeneration of neurons, principally in the striatum, but gradually affecting almost all parts of the brain. This results in a reduction of grey matter and brain atrophy with compensatory enlargement of the lateral brain ventricles.
Nevertheless, also as the second component of the brain parenchyma, the glial cells play an irreplaceable role in this process. The reaction of astrocytes to any damage of the CNS parenchyma in a sense of their conversion into the reactive intensely GFAP-positive subset is well known already for long time.
Although the participation of other types of glial cells, particularly of microglia and NG2 glia, in neurodegenerative process has been studied in last two decades, the histopathological interrelations among all above-mentioned cell types have not been well described yet. Moreover, the validation of existing transgenic rat model of HD51 from this point of view is still lacking. The clinical features of HD described in autopsy records were characteristic for the given stages in all three patients.
However, detailed neurological records or results of genetic testing were not available because old archival material was used. The severity of striatal histopathological changes was graded grades 0—4 according to Vonsattel and coauthors [ 2 ]. Paraffin blocks of brain tissue from autopsies were taken from the neostriatum the caudate nucleus and putamen at the level of the globus pallidus and at the level of the nucleus accumbens. The transcardial perfusion with fixative solution under deep anaesthesia followed by postfixation for 3 days or 2 hours, resp.
After postfixation, the brain hemispheres were separated and processed separately. Histological processing was the same for both the experimental material and autopsies.
Findings obtained by immunofluorescent detections double-labelling were mostly confirmed by a single antibody detection using peroxidase-antiperoxidase PAP immunohistochemistry on parallel paraffin sections. For immunohistochemical detection, deparaffinized and rehydrated sections were used. Sections were then washed and incubated with the appropriate biotinylated secondary antibody Jackson ImmunoResearch Lab.
The sequential technique for immunofluorescent double-labelling of antibodies Ab was same for both types of sections. The negative control, omitting the primary antibody, was made in each labelling. In order to characterize the progression of NDP in the striatum of tgHD51 rats, we used the quantitative analysis of the median diameter of neuronal nuclei as a marker of proposed significant process in a course of neurodegeneration in tgHD rats; it means the shrinkage of striatal neurons.
We would like also to determine the onset of significant neuronal degeneration in the striatum of tgHD51 rats and the possible participation of age-related changes. Selected sections were labelled with NeuN antibody, which marks selectively the nuclei of mature neurons, using the PAP immunohistochemical detection.
The number of analysed sections was the following: Neuronal nucleus median diameter was obtained from 50 independent measurements in the central area of the striatum on each analysed section. Due to possible distortions of the shape of neuronal nucleus in the section, the largest size of the nucleus was considered the nucleus diameter.
Each group of rats of the same age was represented by the set of all medians in given group. Statistical analyses of the differences between groups were performed using MS Excel Microsoft Corp. Surprisingly, the most distinct changes in striatal grey matter develop by the end of the first year of age probably between 9 and 12 months.
The end of the first year represents the turn in the development of morphological changes related to the progression of NDP within the striatum of tgHD51 rats.
These findings correspond to the course of HD in human brain, where the motor and behavioural changes precede the loss of striatal neurons [ 20 ]. We demonstrate possible parallels between the HD progression in humans and the above-described transgenic rat model and prove the validity of our findings for human HD pathology.
The cases demonstrated here represent the sequence of 3 stages grades , grade 3, and grade 4 of the progression of HD in human brain. It is almost impossible to dissociate the alterations referring to neurons and glia in a course of NDP because of very close relationship and mutual influence of both main components of striatal parenchyma.
However, we would like to stress some features specific for each of them in a course of the development of NDP within the striatum of both rat and human brains. For that reason, we described their involvement in progression of NDP separately. When we compare the brains of 2- and 3-month-old, young adult wild-type and tgHD rats, there is no difference in morphology of the striatum. Also lateral brain ventricles are narrow, of the same shape in both mentioned groups Figure 1 a.
Only in month-old tgHD51 rats appears the identifiable enlargement of lateral ventricles, which documents developing striatal atrophy. The process gradually progresses resulting in prominent widening of lateral ventricles Figure 1 b , with concave medial outline of the striatum in the oldest 22—month-old tgHD rats, which is fully comparable with the progression of HD in human brain.
However, with the progression of HD in humans, gradual decrease in number of neurons is significant, particularly in advanced stages of HD grade 3—Figure 4 b and grade 4—Figure 4 c.
Nuclei of striatal neurons are very characteristic, especially due to their large size and fine loosely arranged chromatin in comparison with significantly smaller nuclei with more densely arranged chromatin of glial cell.
Despite the fact that striatal neurons become gradually smaller in course of HD progression compare Figures 3 a and 3 b , such specific features of neuronal and glia nuclei always enable their distinguishing. Our immunohistochemical analysis of neuronal nuclei by NeuN shows slow but already significant progression of neuronal degeneration in the striatum from 12 to 24 months of age of tgHD rats Figure 3 b when compared with age-matched controls Figure 3 a and younger tgHD rats.
The most typical for NDP in tgHD51 rats is a gradual decrease in size of neuronal bodies and nuclei with maintenance of nucleo-cytoplasmic rate , which results in the disintegration and disappearance of affected neurons Figures 2 d — 2 f , ultimately scavenged by microglia Figures 12 b and 12 c.
In the human HD brain, grades with approximately 2-year clinical manifestation , the degeneration and loss of neurons were only random; therefore, the loosening of the neuropil has not been apparent yet. On the other hand, in grade 3 approximately 8-year clinical history , neuronal degeneration was already obvious Figure 4 b.
Depletion of neurons particularly in the CN and putamen Pu accompanied with rarefaction of the neuropil resulted in a reduction of striatal volume and noticeable enlargement of the lateral ventricles.
In grade 4 with approximately year clinical diagnosis the entire corpus striatum CN, Pu, and globus pallidus was affected by degeneration of neurons and neuropil resulting in severe striatal atrophy and therefore the concomitant astrogliosis here prevailed Figures 4 c , 10 b , and 10 c.
The remaining striatal neurons marked by their prominent nuclei gradually became smaller with the progression of NDP, like in the brain of tgHD rats.
Additionally, we confirmed that, alike in human HD brain, neuronal degeneration is selective, that is, affecting primarily certain groups of neurons in the striatum of investigated senescent tgHD rats and moreover that age-related changes contribute to final extent of NDP.
In order to precisely characterize the progression of NDP within the striatum of tgHD51 rats, our morphological findings were supplemented by quantitative analysis of the diameter of neuronal nuclei labelled with NeuN. Also the proportion of age-related changes in this process was assessed. The progression in decrease of the median diameter of neuronal nuclei with age of rats in both wt and tgHD groups of rats is documented by Progress Chart Figure 5 a.
In the first two groups of rats, that is, , and 6-month survivors, no differences in the median diameter of NeuN-positive nuclei were detected when compared within the individual group or among the groups.
Surprisingly, further progression in decrease of the median diameter of neuronal nuclei was not so rapid; however, finally the reduction reached Statistical characteristic of the groups of rats using Box Plot Figure 5 b enables the multiple comparison of the median diameter of neuronal nuclei of the following groups of rats: Differences among three remaining groups are statistically insignificant.
Moreover, it is potentiated with age-related changes particularly in the oldest animals. Unexpectedly, the transitional amelioration of the process up to slight improvement appeared in both groups wt and tgHD of month survivors. Neuronal degeneration in wt rats can be attributed only to the debit of the aging process; the decrease in size of nuclei was slow and the difference between month-old rats and month-old ones was only 8.
Striatal atrophy, in the case of HD, is primarily caused by the degeneration of striatal neurons. Of course, the most prominent feature, seen on histological preparations, is a gradual reduction of neuronal bodies marked by the nuclei. Indeed, the reduction in a volume of neuropil is at least of the same importance.
Although the rarefaction of neuropil is not based only on the degeneration of this network of neuronal processes and synapses, it demonstrates the progression of such process in both human and rat brains Figures 8 a — 8 d. In addition, we also proved the alterations in a character of synapses. In control brains of both rats and humans, synaptophysin-positive synapses are very fine, of uniform size and shape, and plentiful Figures 6 a and 7 a.
With the progression of NDP, most of synapses become coarser, more prominent, but of variable size, and some of them are intensely labelled for synaptophysin; consequently, their number gradually decreases Figures 6 b and 7 b.
Despite different size of synapses in rat Figures 6 a and 6 b and human Figures 7 a and 7 b , the mentioned alterations are of the same character. Since the severity of the striatal damage is also influenced by duration of NDP, the changes in morphology of synapses, and particularly the loosening of neuropil, are certainly more prominent in advanced stages of HD in human brain Figure 7 b than in terminal stage of NDP in tgHD rats Figures 6 b and 8 d.
Additionally, the alterations in glial component and the ageing-associated changes see Section 3. Indeed, the pattern of such process in this basic aspect is the same for both tgHD rats and HD patients. Detection of polyglutamine deposits using polyQ-huntingtin provides interesting findings, which give a complete histopathological picture of HD progression.
In wt rats, polyQ detects a normal polyglutamine domain huntingtin encoded by lower number about 35 or less of consecutive glutamine repeats; therefore, only fine polyQ deposits are spread in the nuclei of striatal neurons Figure 8 a. In contrast, the pathogenic alleles usually contain 39 or more glutamine repeats, which results in production of mhtt and increased density of intranuclear polyQ expression in relation to the progression of NDP of HD phenotype Figures 8 b — 8 d.
Surprisingly, using polyQ-huntingtin antibody, neither typical large intranuclear nor neuropil aggregates were seen. Moreover, neurons in adjacent cortex also exhibit intranuclear but more cytoplasmic polyQ deposits; therefore, they are more densely stained in comparison with striatal neurons, particularly in tgHD rats Figures 8 e — 8 g. On the contrary, only few cortical glial cells express polyQ, which corresponds to the absence of typical reactive gliosis in this region.
It is evident that the developments of changes in glial cell morphology, and certainly also in their function, are conditioned by the intensity and rate of neuronal degeneration in the context of the neuron-glia relationship. Protoplasmic astrocytes are the most numerous component of the striatal parenchyma. The shape of astrocytes changes during the progression of NDP; however specific prominent alterations occur only in human HD brains, where the astrogliosis gradually develops Figures 10 b and 10 c.
Due to only slow development of neuronal degeneration in the striatum of tgHD rats, the subsequent astrogliosis progresses also slowly—with insignificant onset after 6 months of age of tgHD rats—and becomes more distinct just in 18—month-old animals Figure 9 c.
Age-related changes not only are seen in old wt rats, but also participate in progression of reactive gliosis in tgHD rats. Less in wt rats or more in tgHD rats developed striatal atrophy is manifested in senescent rats by denser accumulation of smaller nuclei of neurons and glia compare Figures 2 a — 2 c.
First of all, in both HD patients and tgHD rats, generation of reactive astrocytes proceeds gradually and slowly, unlike the almost immediate appearance of reactive astrocytes after the acute brain damage. Indeed, their bodies are not significantly enlarged hypertrophic ; contrariwise, a part of them also undergoes the degeneration and they are scavenged by microglia Figures 12 c , 13 c , and 13 d.
On the contrary, we never found the reexpression of intermediate filaments nestin and vimentin, which is considerable feature of hypertrophic reactive astrocytes after the acute brain damage. Enactment of respective legislation is pending in several other countries. Using PCR as an analytical method, there is the possibility of a false negative result in cases with a very large CAG repeat expansion. Therefore, labs might put a disclaimer on results where both repeat numbers appear to be the same, indicating that this testing method could rarely miss a very large expansion.
Notably, however, in some studies over one-quarter of HD patients have a negative family history. There are some rare cases with positive family history of neurodegenerative disease, which is a phenocopy of HD lacking the mutation in the HTT gene. Diagnosis may be complicated in rare autoimmune diseases or other syndromes that include choreatic symptoms. The intermediate range between 27 and 35 CAG repeats may disclose a risk of new mutation transmission in offspring, especially when the transmitting parent is a male.
The behavioural abnormalities were discussed as a prodromal stage of HD. Probability not to develop the disease if the test is negative. Assume an increased risk based on family history for a non-affected person. Allelic and locus heterogeneity may need to be considered. Can only be resolved by investigation of the non-affected individual.
Clinical diagnosis of a typical movement disorder in a patient with cognitive and behavioural impairment as well as evidence of typical atrophy in imaging has a high sensitivity in the diagnosis of HD, when the family history is positive for HD.
There are, however, some rare cases of neurodegenerative diseases that can present as phenocopies of HD. Alternate diagnostic procedures are expensive and more time-consuming for the patient, but not harmful.
Alternative diagnostic methods are more expensive and less specific. A positive test result has potential impact on family planning and lifestyle, eg, on the choice of education alternatives, professions, career planning etc.
A negative test result has potential impact on family planning and lifestyle, eg, on the choice of education alternatives, professions, career planning etc. Yes, but invasive prenatal DNA diagnosis is restricted by law in several countries, such as Germany. Pre-implantation diagnosis is technically possible, and it is currently performed, for example, in North America. In both, individuals with negative results potentially due to guilt feelings or positive results concerning the mutation in the HTT gene, the diagnosis can lead to depression and to serious consequences.
Thus, detailed counselling, sufficient time to consider all the potential consequences and psychotherapeutic accompany are necessary as recommended in the predictive testing guidelines. This work was supported by EuroGentest2 Unit 2: JTE declares no conflict of interest.
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Clinical utility gene card for: Huntington's disease
Antibodies. .. Huntington Disease's (HD) is one of the most common inherited genetic diseases that HD is also called Huntington chorea, from the. Experimental models of Huntington's disease. Huntington's disease, also termed Huntington chorea is an inherited .. Chemicals for Western blot. Rats transgenic for Huntington's disease (tgHD51 CAG rats), .. Rarefaction of Neuropil and Alterations in Morphology of Synapses.