Inclusions are sometimes called aggregates or deposits. They are similar in structure to the aggregates found in mad cow disease. It was not known how cell aggregates related to HD. The theories were:
Even non-symptomatic HD carriers have decreased brain blood circulation and that may cause aggregates as a down stream event. --Jerry 10/02/98
From: Cell, Vol. 95, 55–66, October 2, 1998,Frederic Saudou,et al.
The mechanisms by which mutant huntingtin induces neurodegeneration were investigated using a cellular model that recapitulates features of neurodegeneration seen in Huntington’s disease. When transfected into cultured striatal neurons, mutant huntingtin induces neurodegeneration by an apoptotic mechanism. Antiapoptotic compounds or neurotrophic factors protected neurons against mutant huntingtin. Blocking nuclear localization of mutant huntingtin suppressed its ability to form intranuclear inclusions and to induce neurodegeneration. However, the presence of inclusions did not correlate with huntingtin-induced death. The exposure of mutant huntingtin-transfected striatal neurons to conditions that suppress the formation of inclusions resulted in an increase in mutant huntingtin-induced death. These findings suggest that mutant huntingtin acts within the nucleus to induce neurodegeneration. However, intranuclear inclusions may reflect a cellular mechanism to protect against huntingtin-induced cell death.
Huntington’s disease (HD) is a devastating inherited neurodegenerative disease characterized by chorea, personality changes, dementia, and early death (Martin and Gusella, 1986). The characteristic symptoms of patients with HD result from the selective death and dysfunction of specific neuronal subpopulations within the central nervous system. HD leads to significant neuronal death within the striatum, the subcortical brain structure that controls body movements, and to a lesser extent within the cortex (Vonsattel et al., 1985). The specificity of neuronal death seen in HD is striking; within the striatum, the enkephalin-containing medium spiny neurons of the striatum are particularly vulnerable, whereas many of the neighboring neurons remain unaffected (Grave-land et al., 1985; Reiner et al., 1988; Hedreen and Folstein, 1995; Sapp et al., 1995).
Over a decade ago, linkage analysis of families with HD led to the identification of the gene IT-15, whose mutation causes HD (Group THDCR, 1993). The wild-type gene encodes a 350 kDa protein, huntingtin, that bears no homology to known proteins. The huntingtin gene contains a polymorphic stretch of repeated CAG trinucleotides that encodes a polyglutamine (polyGln) tract within huntingtin. When the number of repeats exceeds 35, the gene encodes a version of huntingtin that leads to disease (Gusella et al., 1996).
It has been hypothesized that the expansion of the polyGln tract within huntingtin creates a protein epitope that is distinct from epitopes found in the wild-type huntingtin protein (Trottier et al., 1995a). The new epitope has been theorized to change the interactions between huntingtin and other proteins, thereby leading to neuro-degeneration. If polyGln expansions cause neurodegeneration by rendering huntingtin toxic, it might be expected that all cells that express huntingtin would die, However, the huntingtin protein is expressed widely throughout the central nervous system as well as in in nonneuronal cells, yet only a small subset of these cells die in HD (Gutekunst et al., 1995; Sharp et al., 1995; Trottier et al., 1995b).
As a first step toward understanding huntingtin’s mechanism of action and the basis for its neuronal subtype specificity, recent efforts have been aimed at defining the subcellular site of huntingtin action. In recent studies, huntingtin has been found within nuclei, perikarya, neurites, and synapses (DiFiglia et al., 1995; forma-Gutekunst et al., 1995; Sharp et al., 1995; Trottier et al., 1995b; De Rooij et al., 1996; Saudou et al., 1996). Studies of a transgenic mouse model of HD and of the brains of HD patients have shown that mutant huntingtin can form aggregates within nuclei known as neuronal intranuclear inclusions, suggesting that mutant huntingtin might induce neurodegeneration by acting in the nucleus (Davies et al., 1997; DiFiglia et al., 1997; Becher et al., 1998). The presence of intranuclear inclusions is specific to mutant huntingtin, and inclusions form before neurological symptoms and neurodegeneration occur, suggesting that the aggregation of huntingtin into intranuclear inclusions is a required step in neurodegeneration (Ross, 1997; Davies et al., 1998). However, it has never been shown directly that huntingtin aggregation is toxic; it remains possible that huntingtin aggregation is unrelated to the central mechanisms of neurodegeneration or that neurons promote the aggregation of mutant huntingtin as a protective response against more toxic, soluble forms.
The huntingtin protein may undergo other modifications in addition to aggregation that could be important for its toxic effect. For example, the huntingtin protein contains several consensus sites for the cysteine protease, CPP32 (Goldberg et al., 1996). The observation that N-terminal huntingtin fragments cause disease in mouse transgenic models (Mangiarini et al., 1996) has led to the hypothesis that abnormal polyGln expansions might render huntingtin toxic by facilitating its proteolysis by CPP32 from full-length forms into more toxic N-terminal fragments.
While a mouse model of HD has contributed significantly to the understanding of the mechanisms of Huntingting-induced neurodegeneration (Mangiarini et al., 1996; Davies et al., 1997), cell-based studies represent a complementary approach, which is also likely to provide insight into how huntingtin kills neurons. To characterize the mechanisms of huntingtin-induced neurodegeneration, we have developed a cellular model of HD by introducing huntingtin into cultured striatal neurons by transfection and then assaying the effect of huntingtin expression on neuronal survival. We find that huntingtin is capable of inducing neurodegeneration in a cell-specific manner that depends on the presence of the mutated polyGln stretch. Using this in vitro model of huntingtin-induced neurodegeneration, we find that mutant huntingtin acts within the nucleus to induce neurodegeneration by an apoptotic mechanism. Although mutant huntingtin’s site of action is in the nucleus, the nuclear aggregates of huntingtin are not sufficient to trigger neurodegeneration, and surprisingly their formation is not correlated with huntingtin-induced apoptosis. Rather, conditions that disrupt the formation of intranuclear inclusions lead to an acceleration of huntingtin- induced death. These findings suggest that the formation of intranuclear inclusions may not be a critical step in huntingtin-induced neurodegeneration. We propose that the intranuclear inclusions may instead be part of a protective strategy used by the cell to degrade or inactivate the toxic mutant huntingtin protein.
We have developed a cellular model of Huntington’s disease that has been useful for elucidating mechanisms of huntingtin-induced neurodegeneration and for identifying neuroprotective agents that could be used for the treatment of patients with HD. The expression of huntingtin in cultured striatal neurons causes neurodegeneration by a mechanism that depends specifically on the presence of the mutant polyGln stretch and is cell specific, affecting the same subsets of neurons that degenerate in HD brains. Using this in vitro model, we show that huntingtin induces neurodegeneration by an apoptotic mechanism and that specific inhibitors of apoptosis block huntingtin-induced neurodegeneration. In addition, we have identified two neurotrophic factors, BDNF and CNTF, that protect transfected neurons against huntingtin-induced neuronal death.
We have begun to elucidate the mechanisms of huntingtin-induced neurodegeneration and have found that mutant huntingtin translocates to the nucleus to trigger neuronal apoptosis. This finding is in agreement with studies of transgenic mice expressing mutant ataxin-1 with a mutated NLS, which demonstrate the critical requirement of nuclear localization of mutant ataxin-1 for pathogenesis and aggregate formation in SCA1 (Klement et al., 1998 [this issue of Cell]). Within the nucleus, mutant huntingtin forms inclusions, although the percentage of neurons that display discernible intranuclear inclusions varies depending on the length of the mutant huntingtin fragment that is expressed. Full-length huntingtin forms inclusions very rarely raising the possibility that the intranuclear inclusions may not play a causal role in mutant-huntingtin induced death. This finding is supported by studies of human HD brains in which the number and the location of inclusions do not correlate with the pattern of neurodegeneration (C. A. Gutekunst and S. M. Hersch, personal communication) and by the finding that transgenic mice expressing mutant ataxin-1 in which the self-association domain has been deleted develop a pathological phenotype without forming nuclear aggregates (Klement et al., 1998). Consistent with this possibility is our finding that forms of mutant huntingtin (e.g., full-length mutant huntingtin) that rarely aggregate to form inclusions trigger striatal neuron apoptosis as effectively as N-terminal fragments of mutant huntingtin (e.g., 171–68), which form inclusions quite frequently. It is possible that differences in the ability of full-length mutant huntingtin and the 480–68 and 171–68 huntingtin fragments to form inclusions reflect a difference in the levels of these proteins that accumulate in striatal neurons with the small fragments accumulating to higher levels and therefore forming inclusions more readily. If this proves to be the case it might suggest that lower levels of mutant huntingtin are needed to induce cell death than for intranuclear inclusion formation.
The possibility that intranuclear inclusion formation may not be an important step in the mutant huntingtin-induced death process is corroborated by our finding that hCdc34p(CL->S), a dominant interfering form of an ubiquitin-conjugating enzyme, dramatically suppresses the formation of intranuclear inclusions while accelerating huntingtin-induced death in a CAG expansion dependent manner. Although it is difficult from our analysis to identify the direct site of action of hCdc34p(CL->S), we can conclude that intranuclear inclusion formation can be dissociated from mutant huntingtin induced cell death. Taken together, these findings suggest that huntingtin-containing intranuclear inclusions may form as the cell attempts to degrade or inactivate the toxic mutant huntingtin protein. As such, the formation of intra-nuclear inclusions during mutant huntingtin-induced neurodegeneration may represent a cellular survival strategy in the face of a serious toxic insult.
If nuclear inclusions are not necessary for huntingtin-induced death, how then does huntingtin induce neurodegeneration? We provide evidence that when huntingtin contains a mutated, expanded polyGln stretch, it translocates from its site of synthesis in the cytoplasm into the nucleus where mutant huntingtin accumulates and induces neurodegeneration. The steps that are necessary to translocate mutated huntingtin from the cytoplasm into the nucleus are not known. Full-length mutant huntingtin is sufficiently large that it would have to be actively transported across the nuclear envelope to enter the nucleus. Nuclear import may be mediated by a nuclear localization sequence (NLS) within huntingtin (Bessert et al., 1995). It is possible that nuclear import occurs more readily if mutant full-length huntingtin is first proteolysed into smaller fragments (Goldberg et al., 1996). However, this does not explain why mutant huntingtin, but not wild-type huntingtin, accumulates in the nucleus since both mutant and wild-type huntingtin contain the same NLS sequence. Furthermore, it remains to be shown that proteolysis is required for full-length huntingtin to become toxic and to induce neurodegeneration.
The finding that the addition of an NES to huntingtin blocks neurodegeneration indicates that mutant huntingtin acts in the nucleus and provides insight into the mechanisms of huntingtin-induced neurodegeneration. It is possible that mutant huntingtin exerts its effect in the nucleus through interactions with other proteins or DNA. Nuclear mutant huntingtin could interfere with the DNA binding or activation of transcription factors required for neuronal survival, or mutant huntingtin could itself function within the nucleus as a transcription factor (Gerber et al., 1994), selectively transactivating genes that directly or indirectly regulate apoptosis.
The development of a cellular model should prove
useful for further elucidating mechanisms of huntingtin-induced neurodegeneration and for the identification of new protective factors that could be used for the treatment of patients with HD. Previously, it had been shown that CNTF could prevent neurodegeneration in an excitotoxic model of HD (Emerich et al., 1997), but whether the effects of CNTF or other agents would prove useful in blocking huntingtin-induced neurodegeneration was unknown. The demonstration here that CNTF and BDNF effectively protect neurons against huntingtin-induced death suggests that trophic factor treatments might be a successful strategy for treating HD.Moreover, understanding the signaling mechanisms by which CNTF and BDNF suppress huntingtin-induced
apoptosis could provide additional ways of inhibiting the deleterious effect of the mutant huntingtin protein.
Finally, the NES experiments suggest a completely different strategy for protecting neurons against mutant
huntingtin. If specific molecules within the nuclear envelope translocate mutant huntingtin from the cytoplasm
into the nucleus, then the selective inhibition of mutant huntingtin import might protect neurons against the subsequent
neurodegeneration.